Treating cancer using a telomerase vaccine

ABSTRACT

The invention provides compositions and methods related to human telomerase reverse transcriptase (hTRT), the catalytic protein subunit of human telomerase. The polynucleotides and polypeptides of the invention are useful for diagnosis, prognosis and treatment of human diseases, for changing the proliferative capacity of cells and organisms, and for identification and screening of compounds and treatments useful for treatment of diseases such as cancers.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/843,676, filed Apr. 26, 2001, and a continuation-in part ofU.S. application Ser. No. 10/044,692, filed Jan. 11, 2002, and acontinuation of U.S. patent application Ser. No. 09/432,503, filed Nov.2, 1999, which is a continuation of U.S. patent application Ser. No.08/974,549 filed Nov. 19, 1997, U.S. Pat. No. 6,166,178, which is acontinuation-in-part application of U.S. patent application Ser. No.08/915,503, filed Aug. 14, 1997, abandoned, and a continuation-in-partapplication of U.S. patent application Ser. No. 08/912,951, filed Aug.14, 1997, U.S. Pat. No. 6,475,789 and a continuation-in-part ofapplication of U.S. patent application Ser. No. 08/911,312, filed Aug.14, 1997, abandoned, all three of which are continuations-in-part ofU.S. patent application Ser. No. 08/854,050, filed May 9, 1997, U.S.Pat. No. 6,261,836, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/851,843, filed May 6, 1997, U.S. Pat. No.6,093,809, which is a continuation-in-part of U.S. patent applicationSer. No. 08/846,017, filed Apr. 25, 1997, abandoned, which is acontinuation-in-part of U.S. application Ser. No. 08/844,419 filed Apr.18, 1997, abandoned. This application also claims priority to PatentConvention Treaty Patent Application Serial No.: PCT/US97/17885(published on Apr. 9, 1998 as WO 98/14593) and to Patent ConventionTreaty Patent Application Serial No.: PCT/US97/17618 (published on Apr.9, 1998 as WO 98/14592), both designating the U.S. and filed in the U.S.Receiving Office on Oct. 1, 1997. Each of the aforementionedapplications is explicitly incorporated herein by reference in itsentirety and for all purposes. This application also incorporates byreference copending U.S. patent application Ser. No. 08/974,584, filedNov. 19, 1997, in its entirety and for all purposes.

[0002] This invention was made with Government support under Grant No.GM28039, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention is related to novel nucleic acids encodingthe catalytic subunit of telomerase and related polypeptides. Inparticular, the present invention is directed to the catalytic subunitof human telomerase. The invention provides methods and compositionsrelating to medicine, molecular biology, chemistry, pharmacology, andmedical diagnostic and prognostic technology.

BACKGROUND OF THE INVENTION

[0004] The following discussion is intended to introduce the field ofthe present invention to the reader. The citation of various referencesin this section is not to be construed as an admission of priorinvention.

[0005] It has long been recognized that complete replication of the endsof eukaryotic chromosomes requires specialized cell components (Watson,1972, Nature New Biol., 239:197; Olovnikov, 1973, J Theor. Biol.,41:181). Replication of a linear DNA strand by conventional DNApolymerases requires an RNA primer, and can proceed only 5′ to 3′. Whenthe RNA bound at the extreme 5′ ends of eukaryotic chromosomal DNAstrands is removed, a gap is introduced, leading to a progressiveshortening of daughter strands with each round of replication. Thisshortening of telomeres, the protein-DNA structures physically locatedon the ends of chromosomes, is thought to account for the phenomenon ofcellular senescence or aging (see, e.g., Goldstein, 1990, Science249:1129; Martin et al., 1979, Lab. Invest. 23:86; Goldstein et al.,1969, Proc. Natl. Acad. Sci. USA 64:155; and Schneider and Mitsui, 1976,Proc. Natl. Acad. Sci. USA, 73:3584) of normal human somatic cells invitro and in vivo.

[0006] The length and integrity of telomeres is thus related to entry ofa cell into a senescent stage (i.e., loss of proliferative capacity).Moreover, the ability of a cell to maintain (or increase) telomerelength may allow a cell to escape senescence, i.e., to become immortal.

[0007] The structure of telomeres and telomeric DNA has beeninvestigated in numerous systems (see, e.g, Harley and Villeponteau,1995, Curr. Opin. Genet. Dev. 5:249). In most organisms, telomeric DNAconsists of a tandem array of very simple sequences; in humans and othervertebrates telomeric DNA consists of hundreds to thousands of tandemrepeats of the sequence TTAGGG. Methods for determining and modulatingtelomere length in cells are described in PCT Publications WO 93/23572and WO 96/41016.

[0008] The maintenance of telomeres is a function of a telomere-specificDNA polymerase known as telomerase. Telomerase is a ribonucleoprotein(RNP) that uses a portion of its RNA moiety as a template for telomererepeat DNA synthesis (Morin, 1997, Eur. J. Cancer 33:750; Yu et al.,1990, Nature 344:126; Singer and Gottschling, 1994, Science 266:404;Autexier and Greider, 1994, Genes Develop., 8:563; Gilley et al., 1995,Genes Develop., 9:2214; McEachern and Blackburn, 1995, Nature 367:403;Blackburn, 1992, Ann. Rev. Biochem., 61:113; Greider, 1996, Ann. Rev.Biochem., 65:337). The RNA components of human and other telomeraseshave been cloned and characterized (see, PCT Publication WO 96/01835 andFeng et al., 1995, Science 269:1236). However, the characterization ofthe protein components of telomerase has been difficult. In part, thisis because it has proved difficult to purify the telomerase RNP, whichis present in extremely low levels in cells in which it is expressed.For example, it has been estimated that human cells known to expresshigh levels of telomerase activity may have only about one hundredmolecules of the enzyme per cell.

[0009] Consistent with the relationship of telomeres and telomerase tothe proliferative capacity of a cell (i.e., the ability of the cell todivide indefinitely), telomerase activity is detected in immortal celllines and an extraordinarily diverse set of tumor tissues, but is notdetected (i.e., was absent or below the assay threshold) in normalsomatic cell cultures or normal tissues adjacent to a tumor (see, U.S.Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also,Morin, 1989, Cell 59: 521; Shay and Bacchetti 1997, Eur. J. Cancer33:787; Kim et al., 1994, Science 266:2011; Counter et al., 1992, EMBOJ. 11: 1921; Counter et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91,2900; Counter et al., 1994, J. Virol. 68:3410). Moreover, a correlationbetween the level of telomerase activity in a tumor and the likelyclinical outcome of the patient has been reported (e.g., U.S. Pat. No.5,639,613, supra; Langford et al., 1997, Hum. Pathol. 28:416).Telomerase activity has also been detected in human germ cells,proliferating stem or progenitor cells, and activated lymphocytes. Insomatic stem or progenitor cells, and in activated lymphocytes,telomerase activity is typically either very low or only transientlyexpressed (see, Chiu et al., 1996, Stem Cells 14:239; Bodnar et al.,1996, Exp. Cell Res. 228:58; Taylor et al., 1996, J. Invest. Dermatology106: 759).

[0010] Human telomerase is an ideal target for diagnosing and treatinghuman diseases relating to cellular proliferation and senescence, suchas cancer. Methods for diagnosing and treating cancer and othertelomerase-related diseases in humans are described in U.S. Pat. Nos.5,489,508, 5,639,613, and 5,645,986. Methods for predicting tumorprogression by monitoring telomerase are described in U.S. Pat. No.5,639,613. The discovery and characterization of the catalytic proteinsubunit of human telomerase would provide additional useful assays fortelomerase and for disease diagnosis and therapy. Moreover, cloning anddetermination of the primary sequence of the catalytic protein subunitwould allow more effective therapies for human cancers and otherdiseases related to cell proliferative capacity and senescence.

BRIEF SUMMARY OF THE INVENTION

[0011] The present invention provides an isolated, substantially pure,or recombinant protein preparation of a telomerase reverse transcriptaseprotein, or a variant thereof, or a fragment thereof. In one embodimentthe protein is characterized as having a defined motif that has an aminoacid sequence:

[0012]Trp-R₁-X₇-R₁-R₁-R₂-X-Phe-Phe-Tyr-X-Thr-Glu-X₈₋₉-R₃-R₃-Arg-R₄-X₂-Trp (SEQID NOS:11 and 12)

[0013] where X is any amino acid and a subscript refers to the number ofconsecutive residues, R₁ is leucine or isoleucine, R₂ is glutamine orarginine, R₃ is phenylalanine or tyrosine, and R₄ is lysine orhistidine. In one embodiment the protein has a sequence of human TRT. Inother embodiments, the invention relates to peptides and polypeptidessharing substantial sequence identity with a subsequence of suchproteins.

[0014] In a related embodiment the invention provides an isolated,substantially pure or recombinant nucleic acid that encodes a telomerasereverse transcriptase protein. In one embodiment the nucleic acidencodes a protein comprising an amino acid sequence (SEQ ID NOS:11 and12):

[0015]Trp-R₁-X₇-R₁-R₁-R₂-X-Phe-Phe-Tyr-X-Thr-Glu-X₈₋₉-R₃-R₃-Arg-R₄-X₂-Trp. Inanother embodiment, the nucleic acid has a sequence that encodes thehuman TRT protein. In other embodiments, the invention relates tooligonucleotides and polynucleotides sharing substantial sequenceidentity or complementarity with a subsequence of such nucleic acids.

[0016] In one embodiment, the invention relates to human telomerasereverse transcriptase (hTRT) protein. Thus, in one embodiment, theinvention provides an isolated, substantially pure, or recombinantprotein preparation of an hTRT protein, or a variant thereof, or afragment thereof. In one embodiment, the protein is characterized byhaving an amino acid sequence with at least about 75% or at least about80% sequence identity to the hTRT protein of FIG. 17 (SEQ ID NO:2), or avariant thereof, or a fragment thereof. In a related aspect, the hTRTprotein has the sequence of SEQ ID NO:2. In some embodiments, theprotein has one or more telomerase activities, such as catalyticactivity. In one embodiment, the hTRT protein fragment has at least 6amino acid residues. In other embodiments, the hTRT protein fragment hasat least 8, at least about 10, at least about 12, at least about 15 orat least about 20 contiguous amino acid residues of a naturallyoccurring hTRT polypeptide. In still other embodiments, the hTRT proteinfragment has at least about 50 or at least about 100 amino acidresidues.

[0017] The invention also provides a composition comprising an hTRTprotein and an RNA. The RNA may be a telomerase RNA, such as a humantelomerase RNA. In one embodiment, the hTRT protein and the humantelomerase RNA (hTR) form a ribonucleoprotein complex with a telomeraseactivity.

[0018] In one embodiment, the invention provides isolated humantelomerase comprising hTRT protein, such as a substantially pure humantelomerase comprising hTRT protein and comprising hTR. In oneembodiment, the telomerase is at least about 95% pure. The telomerasemay be isolated from a cell, such as a recombinant host cell in or acell that expresses telomerase activity.

[0019] In another aspect, the invention provides an isolated, synthetic,substantially pure, or recombinant polynucleotide comprising a nucleicacid sequence that encodes an hTRT protein. In one embodiment, thepolynucleotide has a nucleotide sequence encoding an hTRT protein thathas an amino acid sequence as set forth in FIG. 17 (SEQ ID NO:2) or asequence that comprises one or more conservative amino acid (or codon)substitutions or one or more activity-altering amino acid (or codon)substitutions in said amino acid sequence. In a related aspect, thepolynucleotide hybridizes under stringent conditions to a polynucleotidehaving the sequence as set forth in FIG. 16 (SEQ ID NO:1). In anotherrelated aspect, the nucleotide sequence of the polynucleotide has asmallest sum probability of less than about 0.5 when compared to anucleotide sequence as set forth in FIG. 16 (SEQ ID NO:1) using BLASTalgorithm with default parameters.

[0020] In another aspect, the invention provides a polynucleotide havinga promoter sequence operably linked to the sequence encoding the hTRTprotein. The promoter may be a promoter other than the naturallyoccurring hTRT promoter. In a related aspect, the invention provides anexpression vector comprising the promoter of the hTRT.

[0021] The invention also provides an isolated, synthetic, substantiallypure, or recombinant polynucleotide that is at least ten nucleotides inlength and comprises a contiguous sequence of at least ten nucleotidesthat is identical or exactly complementary to a contiguous sequence in anaturally occurring hTRT gene or hTRT mRNA. In some embodiments thepolynucleotide is an RNA, a DNA, or contains one or more non-naturallyoccurring, synthetic nucleotides. In one aspect, the polynucleotide isidentical or exactly complementary to the contiguous sequence of atleast ten contiguous nucleotides in a naturally occurring hTRT gene orhTRT mRNA. For example, the polynucleotide may be an antisensepolynucleotide. In one embodiment, the antisense polynucleotidecomprises at least about 20 nucleotides.

[0022] The invention further provides a method of preparing recombinanttelomerase by contacting a recombinant hTRT protein with a telomeraseRNA component under conditions such that said recombinant protein andsaid telomerase RNA component associate to form a telomerase enzymecapable of catalyzing the addition of nucleotides to a telomerasesubstrate. In one embodiment, the hTRT protein has a sequence as setforth in FIG. 17 (SEQ ID NO:2). The hTRT protein may be produced in anin vitro expression system and mixed with a telomerase RNA or, inanother embodiment, the telomerase RNA can be co-expressed in the invitro expression system. In one embodiment the telomerase RNA is hTR. Inan alternative embodiment, the contacting occurs in a cell, such as ahuman cell. In one embodiment, the cell does not have telomeraseactivity prior to the contacting of the hTRT and the RNA, or theintroduction, such as by transfection, of an hTRT polynucleotide. In oneembodiment, the telomerase RNA is expressed naturally by said cell.

[0023] The invention also provides a cell, such as a human, mouse, oryeast cell, containing the recombinant polynucleotides of the inventionsuch as a polynucleotide with an hTRT protein coding sequence operablylinked a promoter. In particular aspects, the cell is a vertebrate cell,such as a cell from a mammal, for example a human, and has an increasedproliferative capacity relative to a cell that is otherwise identicalbut does not comprise the recombinant polynucleotide or has an increasedtelomerase activity level relative to a cell that is otherwise identicalbut does not comprise the recombinant polynucleotide. In someembodiments the cell is immortal.

[0024] In related embodiments, the invention provides organisms andcells comprising a polynucleotide encoding a human telomerase reversetranscriptase polypeptide, such as a transgenic non-human organism suchas a yeast, plant, bacterium, or a non-human animal, for example, amouse. The invention also provides for transgenic animals and cells fromwhich an hTRT gene has been deleted (knocked-out) or mutated such thatthe gene does not express a naturally occurring hTRT gene product. Thus,in alternative embodiments, the transgenic non-human animal has amutated telomerase gene, is an animal deficient in a telomeraseactivity, is an animal whose TRT deficiency is a result of a mutatedgene encoding a TRT having a reduced level of a telomerase activitycompared to a wild-type TRT and is an animal having a mutated TRT genewith one or more mutations, including missense mutations nonsensemutations, insertions, or deletions.

[0025] The invention also provides an isolated or recombinant antibody,or fragment thereof, that specifically binds to an hTRT protein. In oneembodiment, the antibody binds with an affinity of at least about 10⁸M⁻¹. The antibody may be monoclonal or may be a polyclonal composition,such as a polyclonal antisera. In a related aspect, the inventionprovides a cell capable of secreting the antibody, such as a hybridoma.

[0026] The invention also provides a method for determining whether acompound or treatment is a modulator of a telomerase reversetranscriptase activity or hTRT expression. The method involves detectingor monitoring a change in activity or expression in a cell, animal orcomposition comprising an hTRT protein or polynucleotide followingadministration of the compound or treatment. In one embodiment, themethod includes the steps of providing a TRT composition, contacting theTRT with the test compound and measuring the activity of the TRT, wherea change in TRT activity in the presence of the test compound is anindicator that the test compound modulates TRT activity. In certainembodiments, the composition is a cell, an organism, a transgenicorganism or an in vitro system, such as an expression system, whichcontains a recombinant polynucleotide encoding an hTRT polypeptide.Thus, the hTRT of the method may be a product of in vitro expression. Invarious embodiments the detection of telomerase activity or expressionmay be by detecting a change in abundance of an hTRT gene product,monitoring incorporation of a nucleotide label into a substrate fortelomerase, monitoring hybridization of a probe to an extendedtelomerase substrate, monitoring amplification of an extended telomerasesubstrate, monitoring telomere length of a cell exposed to the testcompound, monitoring the loss of the ability of the telomerase to bindto a chromosome, or measuring the accumulation or loss of telomerestructure.

[0027] In one aspect, the invention provides a method of detecting anhTRT gene product in a biological sample by contacting the biologicalsample with a probe that specifically binds the gene product, whereinthe probe and the gene product form a complex, and detecting thecomplex, where the presence of the complex is correlated with thepresence of the hTRT gene product in the biological sample. The geneproduct may be RNA, DNA or a polypeptide. Examples of probes that may beused for detection include, but are not limited to, nucleic acids andantibodies.

[0028] In one embodiment, the gene product is a nucleic acid which isdetected by amplifying the gene and detecting the amplification product,where the presence of the complex or amplification product is correlatedwith the presence of the hTRT gene product in the biological sample.

[0029] In one embodiment, the biological sample is from a patient, suchas a human patient. In another embodiment the biological sample includesat least one cell from an in vitro cell culture, such as a human cellculture.

[0030] The invention further provides a method of detecting the presenceof at least one immortal or telomerase positive human cell in abiological sample comprising human cells by obtaining the biologicalsample comprising human cells; and detecting the presence in the sampleof a cell having a high level of an hTRT gene product, where thepresence of a cell having a high level of the hTRT gene product iscorrelated with the presence of immortal or telomerase positive cells inthe biological sample.

[0031] The invention also provides a method for diagnosing atelomerase-related condition in a patient by obtaining a cell or tissuesample from the patient, determining the amount of an hTRT gene productin the cell or tissue; and comparing the amount of hTRT gene product inthe cell or tissue with the amount in a healthy cell or tissue of thesame type, where a different amount of hTRT gene product in the samplefrom the patient and the healthy cell or tissue is diagnostic of atelomerase-related condition. In one embodiment the telomerase-relatedcondition is cancer and a greater amount of hTRT gene product isdetected in the sample.

[0032] The invention further provides a method of diagnosing cancer in apatient by obtaining a biological sample from the patient, and detectinga hTRT gene product in the patient sample, where the detection of thehTRT gene product in the sample is correlated with a diagnosis ofcancer.

[0033] The invention further provides a method of diagnosing cancer in apatient by obtaining a patient sample, determining the amount of hTRTgene product in the patient sample; and comparing the amount of hTRTgene product with a normal or control value, where an amount of the hTRTgene product in the patient that is greater than the normal or controlvalue is diagnostic of cancer.

[0034] The invention also provides a method of diagnosing cancer in apatient, by obtaining a patient sample containing at least one cell;determining the amount of an hTRT gene product in a cell in the sample;and comparing the amount of hTRT gene product in the cell with a normalvalue for the cell, wherein an amount of the hTRT gene product greaterthan the normal value is diagnostic of cancer. In one embodiment, thesample is believed to contain at least one malignant cell.

[0035] The invention also provides a method for a prognosing a cancerpatient by determining the amount of hTRT gene product in a cancer cellobtained from the patient; and comparing the amount of hTRT in thecancer cell with a prognostic value of hTRT consistent with a prognosisfor the cancer; where an amount of hTRT in the sample that is at theprognostic value provides the particular prognosis.

[0036] The invention also provides a method for monitoring the abilityof an anticancer treatment to reduce the proliferative capacity ofcancer cells in a patient, by making a first measurement of the amountof an hTRT gene product in at least one cancer cell from the patient;making a second measurement of the level of the hTRT gene product in atleast one cancer cell from the patient, wherein the anticancer treatmentis administered to the patient before the second measurement; andcomparing the first and second measurements, where a lower level of thehTRT gene product in the second measurement is correlated with theability of an anticancer treatment to reduce the proliferative capacityof cancer cells in the patient.

[0037] The invention also provides kits for the detection of an hTRTgene or gene product. In one embodiment, the kit includes a containerincluding a molecule selected from an hTRT nucleic acid or subsequencethereof, an hTRT polypeptide or subsequence thereof, and an anti-hTRTantibody.

[0038] The invention also provides methods of treating human diseases.In one embodiment, the invention provides a method for increasing theproliferative capacity of a vertebrate cell, such as a mammalian cell,by introducing a recombinant polynucleotide into the cell, wherein saidpolynucleotide comprises a sequence encoding an hTRT polypeptide. In oneembodiment, the hTRT polypeptide has a sequence as shown in FIG. 17. Inone embodiment, the sequence is operably linked to a promoter. In oneembodiment, the hTRT has telomerase catalytic activity. In oneembodiment, the cell is human, such as a cell in a human patient. In analternative embodiment, the cell is cultured in vitro. In a relatedembodiment, the cell is introduced into a human patient.

[0039] The invention further provides a method for treating a humandisease by introducing recombinant hTRT polynucleotide into at least onecell in a patient. In one embodiment, a gene therapy vector is used. Ina related embodiment, the method further consists of introducing intothe cell a polynucleotide comprising a sequence encoding hTR, forexample, an hTR polynucleotide operably linked to a promoter.

[0040] The invention also provides a method for increasing theproliferative capacity of a vertebrate cell, said method comprisingintroducing into the cell an effective amount of hTRT polypeptide. Inone embodiment the hTRT polypeptide has telomerase catalytic activity.The invention further provides cells and cell progeny with increasedproliferative capacity.

[0041] The invention also provides a method for treating a conditionassociated with an elevated level of telomerase activity within a cell,comprising introducing into said cell a therapeutically effective amountof an inhibitor of said telomerase activity, wherein said inhibitor isan hTRT polypeptide or an hTRT polynucleotide. In one embodiment, theinhibitor is a polypeptide or polynucleotide comprising, e.g., at leasta subsequence of a sequence shown in FIG. 16, 17, or 20. In additionalembodiments, the polypeptide or polynucleotide inhibits a TRT activity,such as binding of endogenous TRT to telomerase RNA.

[0042] The invention also provides a vaccine comprising an hTRTpolypeptide and an adjuvant. The invention also provides pharmacologicalcompositions containing a pharmaceutically acceptable carrier and amolecule selected from: an hTRT polypeptide, a polynucleotide encodingan hTRT polypeptide, and an hTRT nucleic acid or subsequence thereof.

DESCRIPTION OF THE FIGURES

[0043]FIG. 1 shows highly conserved residues in TRT motifs from human(SEQ ID NO:13), S. pombe (tez1) (SEQ ID NO:14), S. cerevisiae (EST2)(SEQ ID NO:15) and Euplotes aediculatus (p123) (SEQ ID NO:16). Identicalamino acids are indicated with an asterisk (*) [raised slightly], whilethe similar amino acid residues are indicated by a dot (•). Motif “0” inthe figure is also called Motif T; Motif “3” is also called Motif A.

[0044]FIG. 2 shows the location of telomerase-specific and RT-specificsequence motifs of telomerase proteins and other reverse transcriptases.Locations of telomerase-specific motif T and conserved RT motifs 1, 2and A-E are indicated by boxes. The open rectangle labeled HIV-1 RTdelineates the portion of this protein shown in FIG. 3.

[0045]FIG. 3 shows the crystal structure of the p66 subunit of HIV-1reverse transcriptase (Brookhaven code 1HNV). The view is from the backof the right hand to enable all motifs to be shown.

[0046]FIG. 4 shows multiple sequence alignment of telomerase RTs(Sp_Trt1p, S. pombe TRT (SEQ ID NOS:24-29) [also referred to herein as“tezlp”]; hTRT, human TRT (SEQ ID NOS:30-35); Ea_p123, Euplotes p123(SEQ ID NOS:36-41); Sc_Est2p, S. cerevisiae Est2p) (SEQ ID NOS:42-48)and members of other RT families (Sc_al, cytochrome oxidase group IIintron 1-encoded protein from S. cerevisiae mitochondria (SEQ IDNOS:51-56), Dm_TART, reverse transcriptase from Drosophila melanogasterTART non-LTR retrotransposable element) (SEQ ID NOS:57-63; HIV-1, humanimmunodeficiency virus reverse transcriptase (SEQ ID NOS:64-68)). TRTcon (SEQ ID NOS:17-23) and RT con (SEQ ID NOS:49 and 50) representconsensus sequences for telomerase RTs and non-telomerase RTs. Aminoacids are designated with an h, hydrophobic; p, polar; c, charged.Triangles show residues that are conserved among telomerase proteins butdifferent in other RTs. The solid line below motif E highlights theprimer grip region.

[0047]FIG. 5 shows expression of hTRT RNA in telomerase-negative mortalcell strains and telomerase-positive immortal cell lines as described inExample 2.

[0048]FIG. 6 shows a possible phylogenetic tree of telomerases andretroelements rooted with RNA-dependent RNA polymerases.

[0049]FIG. 7 shows a restriction map of lambda clone Gφ5.

[0050]FIG. 8 shows a map of chromosome 5p with the location of the STSmarker D5S678 (located near the hTRT gene) indicated.

[0051]FIG. 9 shows the construction of a hTRT promoter-reporter plasmid.

[0052]FIG. 10, in two pages, shows coexpression in vitro of hTRT and hTRto produce catalytically active human telomerase.

[0053]FIG. 11, in two pages, shows an alignment of sequences from fourTRT proteins from human (hTRT; SEQ ID NOS:72-79), S. pombe Trt1 (spTRT;SEQ ID NOS:80-87), Euplotes p123 (Ea_p123; SEQ ID NOS:88-95), and S.cerevisiae EST2p TRT (Sc_Est2; SEQ ID NOS:96-104) and identifies motifsof interest. TRT con (SEQ ID NOS:69, 21, 70 and 71) shows a TRTconsensus sequence. RT con (SEQ ID NOS:49 and 50) shows consensusresidues for other reverse transcriptases. Consensus residues in uppercase indicate absolute conservation in TRT proteins.

[0054]FIG. 12 shows a Topoisomerase II cleavage site (SEQ ID NO: 108)and NFkB binding site motifs (NFkB_CS1=SEQ ID NO:105; NFkB-MHC-I.2=SEQID NO:106; NFkB_CS2=SEQ ID NO:107) in an hTRT intron, with the sequenceshown corresponding to SEQ ID NO:7.

[0055]FIGS. 13A and 13B show the sequence of the DNA encoding theEuplotes 123 kDa telomerase protein subunit (Euplotes TRT; SEQ IDNO:109).

[0056]FIG. 14 shows the amino acid sequence of the Euplotes 123 kDatelomerase protein subunit (Euplotes TRT protein; SEQ ID NO:110).

[0057]FIGS. 15A-15F show the DNA (SEQ ID NO:111) and amino acid (SEQ IDNO:112) sequences of the S. pombe telomerase catalytic subunit (S. pombeTRT).

[0058]FIG. 16, in two pages, shows the hTRT cDNA sequence, with thesequence shown corresponding to SEQ ID NO:1.

[0059]FIG. 17 shows the hTRT protein encoded by the cDNA of FIG. 16. Theprotein sequence shown corresponds to SEQ ID NO:2.

[0060]FIG. 18 shows the sequence of clone 712562, with the sequenceshown corresponding to SEQ ID NO:3.

[0061]FIG. 19 shows a 259 residue protein encoded by clone 712562, withthe sequence shown corresponding to SEQ ID NO:10.

[0062]FIGS. 20A-20E show the sequence of a nucleic acid with an openreading frame encoding a Δ182 variant polypeptide, with the sequenceshown corresponding to SEQ ID NO:4. This Figure also shows the aminoacid sequence of this Δ182 variant polypeptide, with the amino acidsequence shown corresponding to SEQ ID NO:5.

[0063]FIGS. 21A-21E show sequence from an hTRT genomic clone, with thesequence shown corresponding to SEQ ID NO:6. Consensus motifs andelements are indicated, including sequences characteristic of atopoisomerase II cleavage site, NFκB binding sites, an Alu sequence andother sequence elements.

[0064]FIG. 22 shows the effect of mutation of the TRT gene in yeast, asdescribed in Example 1.

[0065]FIG. 23 shows the sequence of EST AA281296, corresponding to SEQID NO:8.

[0066]FIG. 24 shows the sequence of the 182 basepairs deleted in clone712562, with the sequence shown corresponding to SEQ ID NO:9.

[0067]FIG. 25 shows the results of an assay for telomerase activity fromBJ cells transfected with an expression vector encoding an hTRT protein(PGRN133) or a control plasmid (pBBS212) as described in Example 13.

[0068]FIG. 26 is a schematic diagram of the affinity purification oftelomerase showing the binding and displacement elution steps.

[0069]FIG. 27 is a photograph of a Northern blot of telomerasepreparations obtained during a purification protocol, as described inExample 1. Lane 1 contained 1.5 fmol telomerase RNA, lane 2 contained4.6 fmol telomerase RNA, lane 3 contained 14 fmol telomerase RNA, lane 4contained 41 fmol telomerase RNA, lane 5 contained nuclear extract (42fmol telomerase), lane 6 contained Affi-Gel-heparin-purified telomerase(47 fmol telomerase), lane 7 contained affinity-purified telomerase (68fmol), and lane 8 contained glycerol gradient-purified telomerase (35fmol).

[0070]FIG. 28 shows telomerase activity through a purification protocol.

[0071]FIG. 29 is a photograph of a SDS-PAGE gel, showing the presence ofan approximately 123 kDa polypeptide and an approximately 43 kDa doubletfrom Euplotes aediculatus.

[0072]FIG. 30 is a graph showing the sedimentation coefficient ofEuplotes aediculatus telomerase.

[0073]FIG. 31 is a photograph of a polyacrylamide/urea gel with 36%formamide showing the substrate utilization of Euplotes telomerase.

[0074]FIG. 32 shows the putative alignments of telomerase RNA template(SEQ ID NO:113), and hairpin primers with telomerase RNA.

[0075]FIG. 33 is a photograph of lanes 25-30 of the gel shown in FIG.31, shown at a lighter exposure level (G₄T₄G₄T₄=SEQ ID NO:114).

[0076]FIG. 34 shows the DNA sequence of the gene encoding the 43 kDatelomerase protein subunit from Euplotes (SEQ ID NO:115).

[0077]FIGS. 35A-35D show the DNA sequence (SEQ ID NO:115), as well asthe amino acid sequences of all three open reading frames of the 43 kDatelomerase protein subunit from Euplotes (a=SEQ ID NOS:116-140; b=SEQ IDNOS:141-162; c=SEQ ID NOS:163-186).

[0078]FIGS. 36A and 36B show a sequence comparison between the 123 kDatelomerase protein subunit of Euplotes (SEQ ID NO:187) (upper sequence)and the 80 kDa polypeptide subunit of T. thermophila (SEQ ID NO:188)(lower sequence).

[0079]FIGS. 37A and 37B show a sequence comparison between the 123 kDatelomerase protein subunit of E. aediculatus (SEQ ID NO:189) (uppersequence) and the 95 kDa telomerase polypeptide of T. thermophila (SEQID NO:190) (lower sequence).

[0080]FIG. 38 shows the best-fit alignment between a portion of the“La-domain” of the 43 kDa telomerase protein subunit of E. aediculatus(SEQ ID NO:191) (upper sequence) and a portion of the 95 kDa polypeptidesubunit of T. thermophila (SEQ ID NO:192) (lower sequence).

[0081]FIG. 39 shows the best-fit alignment between a portion of the“La-domain” of the 43 kDa telomerase protein subunit of E. aediculatus(SEQ ID NO:193) (upper sequence) and a portion of the 80 kDa polypeptidesubunit of T. thermophila (SEQ ID NO:194) (lower sequence).

[0082]FIG. 40 shows the alignment and motifs of the polymerase domain ofthe 123 kDa telomerase protein subunit of E. aediculatus (SEQ IDNOS:38-41) and the polymerase domains of various reverse transcriptasesincluding a cytochrome oxidase group II intron 1-encoded protein from S.cerevisiae mitochondria (a1 S.c. (group II)) (SEQ ID NOS:204, 205, 54,206, and 56), Dong (LINE) (SEQ ID NOS:200-203), and yeast ESTp(L8543.12) (SEQ ID NOS:45, 46, 211 and 212), HIV-RT (SEQ ID NOS:207-210)and consensus (SEQ ID NOS:195-199).

[0083]FIG. 41 shows the alignment of a domain of the 43 kDa telomeraseprotein subunit (SEQ ID NO:213) with various La proteins (human La=SEQID NO:214; Xenopus LaA=SEQ ID NO:215; Drosophila La=SEQ ID NO:216; S.c.Lhplp=SEQ ID NO:217).

[0084]FIG. 42 shows the nucleotide sequence encoding the T. thermophila80 kDa protein subunit.

[0085]FIG. 43 shows the amino acid sequence of the T. thermophila 80 kDaprotein subunit (SEQ ID NO:219).

[0086]FIG. 44 shows the nucleotide sequence encoding the T. thermophila95 kDa protein subunit (SEQ ID NO:220).

[0087]FIG. 45 shows the amino acid sequence of the T. thermophila 95 kDaprotein subunit (SEQ ID NO:221).

[0088]FIG. 46 shows the amino acid sequence of L8543.12 (“Est2p”) (SEQID NO:222).

[0089]FIG. 47 shows the alignment of the amino acid sequence encoded bythe Oxytricha PCR product (SEQ ID NO:223) with the Euplotes p123sequence (SEQ ID NO:224).

[0090]FIG. 48 shows the DNA sequence of Est2 (SEQ ID NO:225).

[0091]FIG. 49 shows partial amino acid sequence from a cDNA cloneencoding human telomerase peptide motifs (SEQ ID NO:13).

[0092]FIG. 50 shows partial DNA sequence of a cDNA clone encoding humantelomerase peptide motifs (SEQ ID NO:8).

[0093]FIG. 51 shows the amino acid sequence of tez1, also called S.pombe trt (SEQ ID NO:112).

[0094]FIGS. 52A and 52B show the DNA sequence of tez1 (SEQ ID NO:111).Intronic and other non-coding regions are shown in lower case and exons(i.e., coding regions) are shown in upper case.

[0095]FIG. 53 shows the alignment of EST2p (SEQ ID NO:226), Euplotes(SEQ ID NO:227), and Tetrahymena SEQ ID NO:228) sequences, as well asconsensus sequence (SEQ ID NOS:229-231).

[0096]FIG. 54 shows the sequences of peptides (SEQ ID NOS:232-237)useful for production of anti-hTRT antibodies.

[0097]FIG. 55 is a schematic summary of the tez1⁺ sequencingexperiments.

[0098]FIG. 56 shows two degenerate primers (SEQ ID NOS:238 and 241) usedin PCR to identify the S. pombe homolog of the E. aediculatus p123sequences (SEQ ID NOS:239 and 240).

[0099]FIG. 57 shows the four major bands produced in PCR usingdegenerate primers to identify the S. pombe homolog of the E.aediculatus p123 sequences (SEQ ID NOS:239 and 240).

[0100]FIGS. 58A and 58B show the alignment of the M2 PCR product (SEQ IDNO:243) with E. aediculatus p123 (SEQ ID NO:242), S. cerevisiae (SEQ IDNO:244), and Oxytricha (SEQ ID NO:223) telomerase protein sequences.Also shown are the actual genomic sequences (SEQ ID NOS:246 and 249) andthe peptides encoded (SEQ ID NOS:245 and 250), degenerate primers Poly4(SEQ ID NO:238) and Poly 1 (SEQ ID NO:244), and homologous regions ofthe M2 PCR product (SEQ ID NO:247) and its encoded peptide region (SEQID NO:248).

[0101]FIG. 59 is a schematic showing the 3′ RT PCR strategy foridentifying the S. pombe homolog of the E. aediculatus p123.

[0102]FIG. 60 shows characteristics of the libraries used to screen forS. pombe telomerase protein sequences and shows the results of screeningthe libraries for S. pombe telomerase protein sequences.

[0103]FIG. 61 shows the positive results obtained with theHindIII-digested positive genomic clones containing S. pombe telomerasesequence.

[0104]FIG. 62 is a schematic showing the 5′ RT PCR strategy used toobtain a full length S. pombe TRT clone.

[0105]FIG. 63 shows the alignment of RT domains from telomerasecatalytic subunits for S. pombe (S.p.) (SEQ ID NOS:251-255), S.cerevisiae (S.c.) (SEQ ID NOS:256-260) and E. aediculatus (E.a.) (SEQ IDNOS:261-265). Consensus sequences=SEQ ID NOS:49 and 50.

[0106]FIGS. 64A-64J show the alignment of the sequences from Euplotes(“Ea_p123”) (SEQ ID NO:110), S. cerevisiae (“Sc_Est2p”) (SEQ ID NO:222),and S. pombe (“SP_Tlplp”) (SEQ ID NO:112). In Panel A, the shaded areasindicate residues shared between two sequences. In Panel B, the shadedareas indicate residues shared between all three sequences.

[0107]FIG. 65 shows the disruption strategy used with the telomerasegenes in S. pombe.

[0108]FIG. 66 shows the experimental results confirming disruption oftez1.

[0109]FIG. 67 shows the progressive shortening of telomeres in S. pombedue to tez1 disruption.

[0110]FIGS. 68A-68C show the DNA (SEQ ID NO:266) and amino acid (SEQ IDNO:267) of the ORF encoding an approximately 63 kDa telomerase proteinencoded by the EcoRI-NotI insert of clone 712562.

[0111]FIG. 69 shows an alignment of reverse transcriptase motifs fromvarious sources, E aediculatus p123 (SEQ ID NOS:268-273), S pombe tez1(SEQ ID NOS:274-279), S. cerevisiae EST2 (SEQ ID NOS:280-285), and humanHs TCP1 (SEQ ID NOS:286-291), with various consensus residues and motifsequences (SEQ ID NOS:49 and 50) indicated.

[0112]FIG. 70 provides a restriction and function map of plasmidpGRN121.

[0113]FIGS. 71A and 71B show the results of preliminary nucleic acidsequencing analysis of a hTRT cDNA sequence (SEQ ID NO:292).

[0114]FIGS. 72A-72I show the preliminary nucleic acid sequence of hTRT(SEQ ID NO:292) and deduced ORF sequences in three reading frames (a=SEQID NOS:293-320; b=SEQ ID NOS:321-333; c=SEQ ID NOS:334-342).

[0115]FIG. 73 provides a restriction and function map of plasmidpGRN121.

[0116]FIGS. 74A-74F show refined nucleic acid sequence (SEQ ID NO:343)and deduced ORF sequences (SEQ ID NO:344) of hTRT.

[0117]FIG. 75 shows a restriction map of lambda clone 25-1.1.

DETAILED DESCRIPTION OF THE INVENTION

[0118] I. Introduction

[0119] Telomerase is a ribonucleoprotein complex (RNP) comprising an RNAcomponent and a catalytic protein component. The present inventionrelates to the cloning and characterization of the catalytic proteincomponent of telomerase, hereinafter referred to as “TRT” (telomerasereverse transcriptase). TRT is so named because this protein acts as anRNA-dependent DNA polymerase (reverse transcriptase), using thetelomerase RNA component (hereinafter, “TR”) to direct synthesis oftelomere DNA repeat sequences. Moreover, TRT is evolutionarily relatedto other reverse transcriptases (see Example 12).

[0120] In one aspect, the present invention relates to the cloning andcharacterization of the catalytic protein component of human telomerase,hereinafter referred to as “hTRT.” Human TRT is of extraordinaryinterest and value because, as noted supra, telomerase activity in human(and other mammalian cells) correlates with cell proliferative capacity,cell immortality, and the development of a neoplastic phenotype. Forexample, telomerase activity, and, as demonstrated in Example 2, infra,levels of human TRT gene products and are elevated in immortal humancells (such as malignant tumor cells and immortal cell lines) relativeto mortal cells (such as most human somatic cells).

[0121] The present invention further provides methods and compositionsvaluable for diagnosis, prognosis, and treatment of human diseases anddisease conditions, as described in some detail infra. Also provided aremethods and reagents useful for immortalizing cells (in vivo and exvivo), producing transgenic animals with desirable characteristics, andnumerous other uses, many of which are described infra. The inventionalso provides methods and reagents useful for preparing, cloning, orre-cloning TRT genes and proteins from ciliates, fungi, vertebrates,such as mammals, and other organisms.

[0122] As described in detail infra, TRT was initially characterizedfollowing purification of telomerase from the ciliate Euplotesaediculatus. Extensive purification of E. aediculatus telomerase, usingRNA-affinity chromatography and other methods, yielded the proteinAp123″. Surprisingly, p123 is unrelated to proteins previously believedto constitute the protein subunits of the telomerase holoenzyme (i.e.,the p80 and p95 proteins of Tetrahymena thermophila). Analysis of thep123 DNA and protein sequences (Genbank Accession No. U95964; FIGS. 13and 14) revealed reverse transcriptase (RT) motifs consistent with therole of p123 as the catalytic subunit of telomerase (see, e.g., FIGS. 1,4 and 11). Moreover, p123 is related to a S. cerevisiae (yeast) protein,Est2p, which was known to play a role in maintenance of telomeres in S.cerevisiae (Genbank Accession No. S5396), but prior to the presentinvention was not recognized as encoding a telomerase catalytic subunitprotein (see, e.g., Lendvay et al., 1996, Genetics, 144:1399).

[0123] In one aspect, the present invention provides reagents andmethods for identifying and cloning novel TRTs using: nucleic acidprobes and primers generated or derived from the TRT polynucleotidesdisclosed (e.g., for cloning TRT genes and cDNAs); antibodies thatspecifically recognize the motifs or motif sequences or other TRTepitopes (e.g., for expression cloning TRT genes or purification of TRTproteins); by screening computer databases; or other means. For example,as described in Example 1, PCR (polymerase chain reaction) amplificationof S. pombe DNA was carried out with degenerate-sequence primersdesigned from the Euplotes p123 RT motifs B′ and C. Of four prominentproducts generated, one encoded a peptide sequence homologous toEuplotes p123 and S. cerevisiae Est2p. Using this PCR product as aprobe, the complete sequence of the S. pombe TRT homologue was obtainedby screening of S. pombe cDNA and genomic libraries and amplifying S.pombe RNA by reverse transcription and PCR (RT-PCR). The completesequence of the S. pombe gene (“trt1”; GenBank Accession No. AF015783;FIG. 15) revealed that homology with p123 and Est2p was especially highin the reverse transcriptase motifs. S. pombe trt1 is also referred toas tez1.

[0124] Amplification using degenerate primers derived from thetelomerase RT motifs was also used to obtain TRT gene sequences inOxytricha trifallax and Tetrahymena thermophila, as described in Example1.

[0125] The Euplotes p123, S. pombe trt1, and S. cerevisiae Est2p nucleicacid sequences of the invention were used in a search of a computerizeddatabase of human expressed sequence tags (ESTs) using the program BLAST(Altschul et al. al, 1990, J. Mol. Biol. 215:403). Searching thisdatabase with the Est2p sequence did not indicate a match, but searchingwith p123 and trt1 sequences identified a human EST (Genbank accessionno. AA281296; see SEQ ID NO:8), as described in Example 1, putativelyencoding a homologous protein. Complete sequencing of the cDNA clonecontaining the EST (hereinafter, “clone 712562”; see SEQ ID NO:3) showedthat seven RT motifs were present. However, this clone did not encode acontiguous human TRT with all seven motifs, because motifs B′, C, D, andE were contained in a different open reading frame (ORF) than the moreNH₂-terminal motifs. In addition, the distance between motifs A and B′was substantially shorter than that of the three previouslycharacterized TRTs. Clone 712562 was obtained from the I.M.A.G.E.Consortium; Lennon et al., 1996, Genomics 33:151.

[0126] A cDNA clone, pGRN121, encoding a functional hTRT (see FIG. 16,SEQ ID NO:1) was isolated from a cDNA library derived from the human 293cell line as described in Example 1. Comparing clone 712562 with pGRN121showed that clone 712562 has a 182 base pair (see FIG. 24, SEQ ID NO:9)deletion between motifs A and B. The additional 182 base pairs presentin pGRN121 place all of the TRT motifs in a single open reading frame,and increase the spacing between the motif A and motif B′ regions to adistance consistent with the other known TRTs. As is described infra inthe Examples (e.g., Example 7), SEQ ID NO:1 encodes a catalyticallyactive telomerase protein having the sequence of SEQ ID NO:2. Thepolypeptide of SEQ ID NO:2 has 1132 residues and a calculated molecularweight of about 127 kilodaltons (kD).

[0127] As is discussed infra, and described in Example 9, infra, TRTcDNAs possessing the 182 basepair deletion characteristic of the clone712562 are detected following reverse transcription of RNA fromtelomerase-positive cells (e.g., testis and 293 cells). hTRT RNAslacking this 182 base pair sequence are referred to generally as “Δ182variants” and may represent one, two, or several species. Although thehTRT variants lacking the 182 basepair sequence found in the pGRN121cDNA are unlikely to encode a fully active telomerase catalytic enzyme,they may play a role in telomerase regulation, as discussed infra,and/or have partial telomerase activity, such as telomere binding or hTRbinding activity, as discussed infra.

[0128] Thus, in one aspect, the present invention provides an isolatedpolynucleotide with a sequence of a naturally occurring human TRT geneor mRNA including, but not limited to, a polynucleotide having thesequence as set forth in FIG. 16 (SEQ ID NO:1). In a related aspect, theinvention provides a polynucleotide encoding an hTRT protein, fragment,variant or derivative. In another related aspect, the invention providessense and antisense nucleic acids that bind to an hTRT gene or mRNA. Theinvention further provides hTRT proteins, whether synthesized orpurified from natural sources, as well as antibodies and other agentsthat specifically bind an hTRT protein or a fragment thereof. Thepresent invention also provides many novel methods, including methodsthat employ the aforementioned compositions, for example, by providingdiagnostic and prognostic assays for human diseases, methods fordeveloping therapeutics and methods of therapy, identification oftelomerase-associated proteins, and methods for screening for agentscapable of activating or inhibiting telomerase activity. Numerous otheraspects and embodiments of the invention are provided infra.

[0129] One aspect of the invention is the use of a polynucleotide thatis at least ten nucleotides to about 10 kb or more in length andcomprises a contiguous sequence of at least ten nucleotides that isidentical or exactly complementary to a contiguous sequence in anaturally occurring hTRT gene or hTRT mRNA in assaying or screening foran hTRT gene sequence or hTRT mRNA, or in preparing a recombinant hostcell.

[0130] A further aspect of the invention is the use of an agentincreasing expression of hTRT in the manufacture of a medicament for thetreatment of a condition addressed by increasing proliferative capacityof a vertebrate cell, optionally the medicament being for inhibiting theeffects of aging.

[0131] Yet a further aspect of the invention is the use of an inhibitorof telomerase activity in the manufacture of a medicament for thetreatment of a condition associated with an elevated level of telomeraseactivity within a human cell.

[0132] The proteins, variants and fragments of the invention, and theencoding polynucleotides or fragments, are also each provided in afurther aspect of this invention for use as a pharmaceutical.

[0133] The invention further includes the use of a protein, variant orfragment, or of a polynucleotide or fragment, in each case as definedherein, in the manufacture of a medicament, for example in themanufacture of a medicament for inhibiting an effect of aging or cancer.

[0134] Another aspect of the invention is a polynucleotide selectedfrom:

[0135] (a) the DNA having a sequence as set forth in FIG. 16;

[0136] (b) a polynucleotide of at least 10 nucleotides which hybridizesto the foregoing DNA and which codes for an hTRT protein or variant orwhich hybridizes to a coding sequence for such a variant; and,

[0137] (c) DNA sequences which are degenerate as a result of the geneticcode to the DNA sequences defined in (a) and (b) and which code for anhTRT polypeptide or variant.

[0138] In certain embodiments of the present invention, the hTRTpolynucleotides are other than the 389 nucleotide polynucleotide of SEQID NO:8 and/or other than clone 712562, the plasmid containing aninsert, the sequence of which insert is shown in FIG. 18 (SEQ ID NO:3).

[0139] The description below is organized by topic. Part II furtherdescribes amino acid motifs characteristic of TRT proteins, as well asTRT genes encoding proteins having such motifs. Parts III-VI describe,inter alia, nucleic acids, proteins, antibodies and purifiedcompositions of the invention with particular focus on human TRT relatedcompositions. Part VII describes, inter alia, methods and compositionsof the invention useful for treatment of human disease. Part VIIIdescribes production and identification of immortalized human celllines. Part IX describes, inter alia, uses of the nucleic acids,polynucleotides, and other compositions of the invention for diagnosisof human diseases. Part X describes, inter alia, methods andcompositions of the invention useful for screening and identifyingagents and treatments that modulate (e.g., inhibit or promote)telomerase activity or expression. Part XI describes, inter alia,transgenic animals (e.g., telomerase knockout animals and cells). PartXII is a glossary of terms used in Parts I-XI. Part XIII describesexamples relating to specific embodiments of the invention. Theorganization of the description of the invention by topic and subtopicis to provide clarity, and not to be limiting in any way.

[0140] II. TRT Genes and Proteins

[0141] The present invention provides isolated and/or recombinant genesand proteins having a sequence of a telomerase catalytic subunit protein(i.e., telomerase reverse transcriptase), including, but not limited to,the naturally occurring forms of such genes and proteins in isolated orrecombinant form. Typically, TRTs are large, basic, proteins havingreverse transcriptase (RT) and telomerase-specific (T) amino acidmotifs, as disclosed herein. Because these motifs are conserved acrossdiverse organisms, TRT genes of numerous organisms may be obtained usingthe methods of the invention or identified using primers, nucleic acidprobes, and antibodies of the invention, such as those specific for oneor more of the motif sequences.

[0142] The seven RT motifs found in TRTs, while similar to those foundin other reverse transcriptases, have particular hallmarks. For example,as shown in FIG. 4, within the TRT RT motifs there are a number of aminoacid substitutions (marked with arrows) in residues highly conservedamong the other RTs. For example, in motif C the two aspartic acidresidues (DD) that coordinate active site metal ions (see, Kohlstaedt etal., 1992, Science 256:1783; Jacobo-Molina et al., 1993, Proc. Natl.Acad. Sci. U.S.A. 90:6320; Patel et al., 1995, Biochemistry 34:5351)occur in the context hxDD(F/Y) (SEQ ID NO:345) in the telomerase RTscompared to (F/Y)xDDh (SEQ ID NO:346) in the other RTs (where “h” is ahydrophobic amino acid, and “x” is any amino acid; see Xiong et al.,1990, EMBO J. 9:3353; Eickbush, in The Evolutionary Biology of Viruses,(S. Morse, Ed., Raven Press, NY, p. 121, 1994)). Another systematicchange characteristic of the telomerase subgroup occurs in motif E,where WxGxSx (SEQ ID NO:347) is a consensus sequence or is conservedamong the telomerase proteins, whereas hLGxxh (SEQ ID NO:348) ischaracteristic of other RTs (Xiong et al., supra; Eickbush supra). Thismotif E is called the “primer grip”, and mutations in this region havebeen reported to affect RNA priming but not DNA priming (Powell et al.,1997, J. Biol. Chem. 272:13262). Because telomerase requires a DNAprimer (e.g., the chromosome 3′ end), it is not unexpected thattelomerase should differ from other RTs in the primer grip region. Inaddition, the distance between motifs A and B′ is longer in the TRTsthan is typical for other RTs, which may represent an insertion withinthe “fingers” region of the structure which resembles a right hand (FIG.3; see Kohlstaedt et al., supra; Jacobo-Molina et al., supra; and Patelet al., supra).

[0143] Moreover, as noted supra, Motif T is an additional hallmark ofTRT proteins. This Motif T, as shown, for example in FIG. 4(W-L-X-Y-X-X-h-h-X-h-h-X-p-F-F-Y-X-T-E-X-p-X-X-X-p-X-X-X-Y-X-R-K-X-X-W(SEQ ID NO:349) [X is any amino acid, h is hydrophobic, p is polar]),comprises a sequence that can be described using the formula:Trp-R₁-X₇-R₁-R₁-R₂-X-Phe- (SEQ ID NOS:11 and 12)Phe-Tyr-X-Thr-Glu-X₈₋₉-R₃- R₃-Arg-R₄-X₂-Trp

[0144] where X is any amino acid and the subscript refers to the numberof consecutive residues, R₁ is leucine or isoleucine, R₂ is glutamine orarginine, R₃ is phenylalanine or tyrosine, and R₄ is lysine orhistidine.

[0145] The T motif can also be described using the formula:Trp-R₁-X₄-h-h-X-h-h-R₂-p- (SEQ ID NOS:350 and 351)Phe-Phe-Tyr-X-Thr-Glu-X-p- X₃-p-X₂₋₃-R₃-R₃-Arg-R₄-X₂- Trp

[0146] where X is any amino acid and a subscript refers to the number ofconsecutive residues, R₁ is leucine or isoleucine, R₂ is glutamine orarginine, R₃ is phenylalanine or tyrosine, R₄ is lysine or histidine, his a hydrophobic amino acid selected from Ala, Leu, Ile, Val, Pro, Phe,Trp, and Met, and p is a polar amino acid selected from Gly, Ser, Thr,Tyr, Cys, Asn and Gln.

[0147] In one embodiment, the present invention provides isolatednaturally occurring and recombinant TRT proteins comprising one or moreof the motifs illustrated in FIG. 11, e.g., Motif T W-X₁₂-FFY-X-TE- (SEQID NOS:352 X₁₀₋₁₁-R-X₃-W-X₇-I and 353) Motif T′ E-X₂-V-X (SEQ ID NO:354)Motif 1 X₃-R-X₂-P-K-X₃, (SEQ ID NO:355) or, alternatively, h-R-h-X-P-K(SEQ ID NO:633) Motif 2 X-R-X-I-X (SEQ ID NO:356) or, alternatively,(F/L)-R-h-I-X₂-h (SEQ ID NO:634) Motif A X₄-F-X₃-D-X₄-YD- (SEQ IDNO:357) X₂, or, alternatively, P-X-L-Y-F-h-X-h-D- (SEQ ID NO:635)h-X₂-C-Y-D-X-I Motif B′ Y-X₄-G-X₂-QG-X₃-S- (SEQ ID NO:358) X₈ or,alternatively, K-X-Y-X-Q-X₂-G-I-P- (SEQ ID NO:636) Q-G-S-X-L-S-X-h-LMotif C X₆-DD-X-L-X₃, (SEQ ID NO:359) or, alternatively,L-L-R-L-X-D-D-X-L- (SEQ ID NO:637) h-I-T

[0148] When the TRT protein shown contains more than one TRT motif, theorder (NH2->COOH) is as shown in FIG. 11.

[0149] In one embodiment, the present invention provides isolatednaturally occurring TRT proteins comprising the following supermotif:(NH₂)-X₃₀₀₋₆₀₀-W-X₁₂-FFY-X-TE- (SEQ ID NO:727)X₁₀₋₁₁-R-X₃-W-X₇-I-X₅₋₂₀-E-X₂-V-X- X₅₋₂₀-X₃-R-X₂-PK-X₄₋₁₀-R-X-I-X-X₆₀₋₈₀-X₄-F-X₃-D-X₄-YD-X₂-X₈₀₋₁₃₀- Y-X₄-G-X₂-QG-X₃-S-X₈-X₅₋₃₅-X₆-DD-X-L-X₃-X₁₀₋₂₀-X₁₂-K

[0150] It will be apparent to one of skill that, provided with thereagents, including the TRT sequences disclosed herein for thosereagents and the methods and guidance provided herein (includingspecific methodologies described infra), TRT genes and proteins can beobtained, isolated and produced in recombinant form by one of ordinaryskill. For example, primers (e.g., degenerate amplification primers) areprovided that hybridize to gene sequences encoding RT and T motifscharacteristic of TRT. For example, one or more primers or degenerateprimers that hybridize to sequences encoding the FFYXTE (SEQ ID NO:360)region of the T motif, other TRT motifs (as discussed infra), orcombinations of motifs or consensus sequences, can be prepared based onthe codon usage of the target organism, and used to amplify the TRT genesequence from genomic DNA or cDNA prepared from the target organism. Useof degenerate primers is well known in the art and entails use of setsof primers that hybridize to the set of nucleic acid sequences that canpotentially encode the amino acids of the target motif, taking intoaccount codon preferences and usage of the target organism, and by usingamplification (e.g., PCR) conditions appropriate for allowing basemismatches in the annealing steps of PCR. Typically two primer sets areused; however, single primer (or, in this case, a single degenerateprimer set) amplification systems are well known and may be used toobtain TRT genes.

[0151] Table 1 provides illustrative primers of the invention that maybe used to amplify novel TRT nucleic acids, particularly those fromvertebrates (e.g., humans and other mammals). “N” is an equimolarmixture of all four nucleotides, and nucleotides within parentheses areequimolar mixtures of the specified nucleotides. TABLE 1 ILLUSTRATIVEDEGENERATE PRIMERS FOR AMPLIFICATION OF TRT NUCLEIC ACIDS motif primermotif SEQ ID NO: direction 5′ sequence-3′ SEQ ID NO: a FFYPVTE 361Forward TT(CT)TT(CT)TA(CT)GTNACNGA 362 b FFYVTE 361 ReverseTGNGTNAC(GA)TA(GA)AA(GA)AA 363 c RFIPKP 364 Forward(CA)GNTT(CT)AT(ACT)CCNAA(AG)CC 365 d RFIPKP 364 ReverseGG(TC)TTNGG(TGA)AT(GA)AANC 366 e AYDTI 367 Forward GCNTA(CT)GA(CT)ACNAT368 f AYDTI 367 Reverse TANGT(GA)TC(GA)TANGC 369 g GIPOG 370 ForwardGGNAT(ACT)CCNCA(AG)GG 371 h GIPOGS 21 Reverse(GC)(AT)NCC(TC)TGNGG(TGA)ATNCC 372 i LVDDFL 373 Forward(CT)TNGTNGA(CT)GA(CT)TT(CT)(CT)T 374 j DDFLLVT 375 ReverseGTNACNA(GA)NA(GA)(GA)AA(GA)TG(GA)TC 376

[0152] Preferred primer combinations (y=yes, n=no) Reverse Forward b d fh j a n y y y y c n n y y y e n n n y y g n n n n y i n n n n n

[0153] In one embodiment, an amplified TRT nucleic acid is used as ahybridization probe for colony hybridization to a library (e.g., cDNAlibrary) made from the target organism, such that a nucleic acid havingthe entire TRT protein coding sequence, or a substantial portionthereof, is identified and isolated or cloned. Reagents and methods suchas those just described were used in accordance with the methodsdescribed herein to obtain TRT gene sequences of Oxytricha trifallax andTetrahymena thermophila, as described in detail infra. It will berecognized that following cloning of a previously uncharacterized TRTgene, the sequence can be determined by routine methods and the encodedpolypeptide synthesized and assayed for a TRT activity, such astelomerase catalytic activity (as described herein and/or by telomeraseassays known in the art).

[0154] It will also be apparent to those of skill that TRT genes may becloned using any of a variety of cloning methods of the inventionbecause the TRT motif sequences and the nucleic acids of the inventioncomprising such sequences can be used in a wide variety of such methods.For example, hybridization using a probe based on the sequence of aknown TRT to DNA or other nucleic acid libraries from the targetorganism, as described in Example 1 can be used. It will be appreciatedthat degenerate PCR primers or their amplification products such asthose described supra, may themselves be labeled and used ashybridization probes. In another embodiment, expression cloning methodsare used. For example, one or more antibodies that specifically bindpeptides that span a TRT motif or other TRT epitope, such as the FFYXTE(SEQ ID NO:360) motif can be employed to isolate a ribosomal complexcomprising a TRT protein and the mRNA that encodes it. For generatingsuch antibodies of the invention, the peptide immunogens are typicallybetween 6 and 30 amino acids in length, more often about 10 to 20 aminoacids in length. The antibodies may also be used to probe a cDNAexpression library derived from the organism of interest to identify aclone encoding a TRT sequence. In another embodiment, computer searchesof DNA databases for DNAs containing sequences conserved with known TRTscan also be used to identify a clone comprising TRT sequence.

[0155] In one aspect, the present invention provides compositionscomprising an isolated or recombinant polypeptide having the amino acidsequence of a naturally occurring TRT protein. Usually the naturallyoccurring TRT has a molecular weight of between about 80,000 daltons (D)and about 150,000 D, most often between about 95,000 D and about 130,000D. Typically, the naturally occurring TRT has a net positive charge atpH 7 (calculated pI typically greater than 9). In one embodiment, thepolypeptide exhibits a telomerase activity as defined herein. In arelated embodiment, the polypeptide has a TRT-specific region (T motif)sequence and exhibits a telomerase activity. The invention furtherprovides fragments of such polypeptides. The present invention alsoprovides isolated or recombinant polynucleotide having the sequence of anaturally occurring gene encoding a TRT protein. The invention providesregents useful for isolating sequence of a TRT from nonvertebrate (suchas a yeast) and vertebrates, such as mammals (e.g., murine or human).The isolated polynucleotide may be associated with other naturallyoccurring or recombinant or synthetic vector nucleic acid sequences.Typically, the isolated nucleic acid is smaller than about 300 kb, oftenless than about 50 kb, more often less than about 20 kb, frequently lessthan about 10 kb and sometimes less than about 5 kb or 2 kb in length.In some embodiments the isolated TRT polynucleotide is even smaller,such as a gene fragment, primer, or probe of less than about 1 kb orless than 0.1 kb.

[0156] III. Nucleic Acids

[0157] A) Generally

[0158] The present invention provides isolated and recombinant nucleicacids having a sequence of a polynucleotide encoding a telomerasecatalytic subunit protein (TRT), such as a recombinant TRT gene fromEuplotes, Tetrahymena, S. pombe or humans. Exemplary polynucleotides areprovided in FIG. 13 (Euplotes); FIG. 15 (S. pombe) and FIG. 16 (human,GenBank Accession No. AF015950). The present invention provides senseand anti-sense polynucleotides having a TRT gene sequence, includingprobes, primers, TRT-protein-encoding polynucleotides, and the like.

[0159] B) Human TRT

[0160] The present invention provides nucleic acids having a sequence ofa telomerase catalytic subunit from humans (i.e., hTRT).

[0161] In one aspect, the invention provides a polynucleotide having asequence or subsequence of a human TRT gene or RNA. In one embodiment,the polynucleotide of the invention has a sequence of SEQ ID NO: 1 shownin FIG. 16 or a subsequence thereof. In another embodiment, thepolynucleotide has a sequence of SEQ ID NO:3 (FIG. 18), SEQ ID NO:4(FIG. 20), or subsequences thereof. The invention also providespolynucleotides with substantial sequence identity to the hTRT nucleicacid sequences disclosed herein, e.g., including but not limited to SEQID NOS:1 [FIG. 16], 4 [FIG. 20], 6 [FIG. 21], and 7 [FIG. 12]). Thus,the invention provides naturally occurring alleles of human TRT genesand variant polynucleotide sequences having one or more nucleotidedeletions, insertions or substitutions relative to an hTRT nucleic acidsequence disclosed herein. As described infra, variant nucleic acids maybe produced using the recombinant or synthetic methods described belowor by other means.

[0162] The invention also provides isolated and recombinantpolynucleotides having a sequence from a flanking region of a human TRTgene. Such polynucleotides include those derived from genomic sequencesof untranslated regions of the hTRT mRNA. An exemplary genomic sequenceis shown in FIG. 21 (SEQ ID NO:6). As described in Example 4, SEQ IDNO:6 was obtained by sequencing a clone, λGΦ5 isolated from a humangenomic library. Lambda GΦ5 contains a 15 kilobasepair (kbp) insertincluding approximately 13,000 bases 5′ to the hTRT coding sequences.This clone contains hTRT promoter sequences and other hTRT generegulatory sequences (e.g., enhancers).

[0163] The invention also provides isolated and recombinantpolynucleotides having a sequence from an intronic region of a human TRTgene. An exemplary intronic sequence is shown in FIG. 12 (SEQ ID NO: 7;see Example 3). In some embodiments, hTRT introns are included in“minigenes” for improved expression of hTRT proteins in eukaryoticcells.

[0164] In a related aspect, the present invention providespolynucleotides that encode hTRT proteins or protein fragments,including modified, altered and variant hTRT polypeptides. In oneembodiment, the encoded hTRT protein or fragment has an amino acidsequence as set forth in FIG. 17 (SEQ ID NO:2), or with conservativesubstitutions of SEQ ID NO:2. In one embodiment, the encoded hTRTprotein or fragment has substitutions that change an activity of theprotein (e.g., telomerase catalytic activity).

[0165] It will be appreciated that, as a result of the degeneracy of thegenetic code, the nucleic acid encoding the hTRT protein need not havethe sequence of a naturally occurring hTRT gene, but that a multitude ofpolynucleotides can encode an hTRT polypeptide having an amino acidsequence of SEQ ID NO:2. The present invention provides each and everypossible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices made inaccordance with known triplet genetic codes, and all such variations arespecifically disclosed hereby. Thus, although in some cases hTRTpolypeptide-encoding nucleotide sequences that are capable ofhybridizing to the nucleotide sequence of the naturally occurringsequence (under appropriately selected conditions of stringency) arepreferred, it may be advantageous in other cases to produce nucleotidesequences encoding hTRT that employ a substantially different codonusage and so perhaps do not hybridize to nucleic acids with thenaturally occurring sequence.

[0166] In particular embodiments, the invention provides hTRT oligo- andpolynucleotides that comprise a subsequence of an hTRT nucleic aciddisclosed herein (e.g., SEQ ID NOS:1 and 6). The nucleic acids of theinvention typically comprise at least about 10, more often at leastabout 12 or about 15 consecutive bases of the exemplified hTRTpolynucleotide. Often, the nucleic acid of the invention will comprise alonger sequence, such as at least about 25, about 50, about 100, about200, or at least about 500 to 3000 bases in length, for example whenexpression of a polypeptide, or full length hTRT protein is intended.

[0167] In still other embodiments, the present invention provides “Δ182Htrt” polynucleotides having a sequence identical or complementary tonaturally occurring or non-naturally occurring hTRT polynucleotides suchas SEQ ID NO:3 or SEQ ID NO:4, which do not contain the 182 nucleotidesequence (SEQ ID NO:9) found in pGRN121 (and also absent in clone712562). These polynucleotides are of interest, in part, because theyencode polypeptides that contain different combinations or arrangementsof TRT motifs than found in the “full-length” hTRT polypeptide (SEQ IDNO:2) such as is encoded by pGRN121. As discussed infra, it iscontemplated that these polypeptides may play a biological role innature (e.g., in regulation of telomerase expression in cells) and/orfind use as therapeutics (e.g., as dominant-negative products thatinhibit function of wild-type proteins), or have other roles and uses,e.g. as described herein.

[0168] For example, in contrast to the polypeptide encoded by pGRN121,clone 712562 encodes a 259 residue protein with a calculated molecularweight of approximately 30 kD (hereinafter, “712562 hTRT”). The 712562hTRT polypeptide (SEQ ID NO:10 [FIG. 19]) contains motifs T, 1, 2, andA, but not motifs B′, C, D and E (See FIG. 4). Similarly, a variant hTRTpolypeptide with therapeutic and other activities may be expressed froma nucleic acid similar to the pGRN121 cDNA but lacking the 182 basepairsmissing in clone 712562, e.g., having the sequence shown in FIG. 20 (SEQID NO:4). This nucleic acid (hereinafter, “pro90 hTRT”), which may besynthesized using routine synthetic or recombinant methods as describedherein, encodes a protein of 807 residues (calculated molecular weightof approximately 90 kD) that shares the same amino terminal sequence asthe hTRT protein encoded by SEQ ID NO:1, but diverges at thecarboxy-terminal region (the first 763 residues are common, the last 44residues of pro90 hTRT are different than “full-length” hTRT). The pro90hTRT polypeptide contains motifs T, 1, 2, and A, but not motifs B, C, D,E, and thus may have some, but not likely all telomerase activities.

[0169] C) Production of Human TRT Nucleic Acids

[0170] The polynucleotides of the invention have numerous usesincluding, but not limited to, expression of polypeptides encoding hTRTor fragments thereof, use as sense or antisense probes or primers forhybridization and/or amplification of naturally occurring hTRT genes orRNAs (e.g. for diagnostic or prognostic applications), and astherapeutic agents (e.g., in antisense, triplex, or ribozymecompositions). As will be apparent upon review of the disclosure, theseuses will have enormous impact on the diagnosis and treatment of humandiseases relating to aging, cancer, and fertility as well as the growth,reproduction, and manufacture of cell-based products. As described inthe following sections, the hTRT nucleic acids of the invention may bemade (e.g., cloned, synthesized, or amplified) using techniques wellknown in the art.

[0171] 1) Cloning, Amplification, and Recombinant Production

[0172] In one embodiment, hTRT genes or cDNAs are cloned using a nucleicacid probe that specifically hybridizes to an hTRT mRNA, cDNA, orgenomic DNA. One suitable probe for this purpose is a polynucleotidehaving all or part of the sequence provided in FIG. 16 (SEQ ID NO:1),such as a probe comprising a subsequence thereof. Typically, the targethTRT genomic DNA or cDNA is ligated into a vector (e.g., a plasmid,phage, virus, yeast artificial chromosome, or the like) and may beisolated from a genomic or cDNA library (e.g., a human placental cDNAlibrary). Once an hTRT nucleic acid is identified, it can be isolatedaccording to standard methods known to those of skill in the art. Anillustrative example of screening a human cDNA library for the hTRT geneis provided in Example 1; similarly, an example of screening a humangenomic library is found in Examples 3 and 4. Cloning methods are wellknown and are described, for example, in Sambrook et al., (1989)MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold SpringHarbor Laboratory hereinafter, “Sambrook”); Berger and Kimmel, (1987)METHODS IN ENZYMOLOGY, VOL.152: GUIDE TO MOLECULAR CLONING TECHNIQUES,San Diego: Academic Press, Inc.; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, New York(1997); Cashion et al., U.S. Pat. No. 5,017,478; and Carr, EuropeanPatent No. 0,246,864.

[0173] The invention also provides hTRT genomic or cDNA nucleic acidsisolated by amplification methods such as the polymerase chain reaction(PCR). In one embodiment, hTRT protein coding sequence is amplified froman RNA or cDNA sample (e.g., double stranded placental cDNA (Clontech,Palo Alto Calif.)) using the primers 5′-GTGAAGGCACTGTTCAGCG-3′(“TCP1.1”) (SEQ ID NO:377) and 5′-CGCGTGGGTGAGGTGAGGTG-3 (“TCP 1.15”)(SEQ ID NO:378). In some embodiments a third primer or second pair ofprimers may be used, e.g., for “nested PCR”, to increase specificity.One example of a second pair of primers is 5′-CTGTGCTGGGCCTGGACGATA-3′(“TCP1.14”) (SEQ ID NO:379) and 5′-AGCTTGTTCTCCATGTCGCCGTAG-3′(“billTCP6”) (SEQ ID NO:380). It will be apparent to those of skill thatnumerous other primers and primer combinations, useful for amplificationof hTRT nucleic acids are provided by the present invention.

[0174] Moreover, the invention provides primers that amplify anyspecific region (e.g., coding regions, promoter regions, and/or introns)or subsequence of hTRT genomic DNA, cDNA or RNA. For example, the hTRTintron at position 274/275 of SEQ ID NO:1 (see Example 3) may beamplified (e.g., for detection of genomic clones) using primers TCP1.57and TCP1.52 (primer pair 1) or primers TCP1.49 and TCP1.50 (primer pair2). (Primer names refer to primers listed in Table 2, infra.) The primerpairs can be used individually or in a nested PCR where primer set 1 isused first. Another illustrative example relates to primers thatspecifically amplify and so detect the 5′ end of the hTRT mRNA or theexon encoding the 5′ end of hTRT gene (e.g., to assess the size orcompleteness of a cDNA clone). The following primer pairs are useful foramplifying the 5′ end of hTRT: primers K320 and K321 (primer pair 3);primers K320 and TCP1.61 (primer pair 4); primers K320 and K322 (primerpair 5). The primer sets can be used in a nested PCR in the order set 5,then set 4 or set 3, or set 4 or set 5, then set 3. Yet anotherillustrative example involves primers chosen to amplify or detectspecifically the conserved hTRT TRT motif region comprisingapproximately the middle third of the mRNA (e.g., for use as ahybridization probe to identify TRT clones from, for example, nonhumanorganisms). The following primer pairs are useful for amplifying the TRTmotif region of hTRT nucleic acids: primers K304 and TCP1.8 (primer pair6), or primers Lt1 and TCP1.15 (primer pair 7). The primer sets can beused in a nested PCR experiment in the order set 6 then set 7.

[0175] Suitable PCR amplification conditions are known to those of skilland include (but are not limited to) 1 unit Taq polymerase (PerkinElmer, Norwalk Conn.), 100 μM each dNTP (dATP, dCTP, dGTP, dTTP), 1×PCRbuffer (50 mM KCl, 10 mM Tris, pH 8.3 at room temperature, 1.5 mM MgCl₂,0.01% gelatin) and 0.5 μM primers, with the amplification run for about30 cycles at 94° for 45 sec, 55° for 45 sec and 72° for 90 sec. It willbe recognized by those of skill in the art that other thermostable DNApolymerases, reaction conditions, and cycling parameters will alsoprovide suitable amplification. Other suitable in vitro amplificationmethods that can be used to obtain hTRT nucleic acids include, but arenot limited to, those herein, infra. Once amplified, the hTRT nucleicacids can be cloned, if desired, into any of a variety of vectors usingroutine molecular biological methods or detected or otherwise utilizedin accordance with the methods of the invention.

[0176] One of skill will appreciate that the cloned or amplified hTRTnucleic acids obtained as described above can be prepared or propagatedusing other methods, such as chemical synthesis or replication bytransformation into bacterial systems, such as E. coli (see, e.g.,Ausubel et al., supra), or eukaryotic, such as mammalian, expressionsystems. Similarly, hTRT RNA can be expressed in accordance with thepresent in vitro methods, or in bacterial systems such as E. coli using,for example, commercially available vectors containing promotersrecognized by an RNA polymerase such as T7, T3 or SP6, or transcriptionof DNA generated by PCR amplification using primers containing an RNApolymerase promoter.

[0177] The present invention further provides altered or modified hTRTnucleic acids. It will be recognized by one of skill that the cloned oramplified hTRT nucleic acids obtained can be modified (e.g., truncated,derivatized, altered) by methods well known in the art (e.g.,site-directed mutagenesis, linker scanning mutagenesis) or simplysynthesized de novo as described below. The altered or modified hTRTnucleic acids are useful for a variety of applications, including, butnot limited to, facilitating cloning or manipulation of an hTRT gene orgene product, or expressing a variant hTRT gene product. For example, inone embodiment, the hTRT gene sequence is altered such that it encodesan hTRT polypeptide with altered properties or activities, as discussedin detail in infra, for example, by mutation in a conserved motif ofhTRT. In another illustrative example, the mutations in the proteincoding region of an hTRT nucleic acid may be introduced to alterglycosylation patterns, to change codon preference, to produce splicevariants, remove protease-sensitive sites, create antigenic domains,modify specific activity, and the like. In other embodiments, thenucleotide sequence encoding hTRT and its derivatives is changed withoutaltering the encoded amino acid sequences, for example, the productionof RNA transcripts having more desirable properties, such as increasedtranslation efficiency or a greater or a shorter half-life, compared totranscripts produced from the naturally occurring sequence. In yetanother embodiment, altered codons are selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Useful in vitro and in vivorecombinant techniques that can be used to prepare variant hTRTpolynucleotides of the invention are found in Sambrook et al. andAusubel et al., both supra.

[0178] As noted supra, the present invention provides nucleic acidshaving flanking (5′ or 3′) and intronic sequences of the hTRT gene. Thenucleic acids are of interest, inter alia, because they contain promoterand other regulatory elements involved in hTRT regulation and useful forexpression of hTRT and other recombinant proteins or RNA gene products.It will be apparent that, in addition to the nucleic acid sequencesprovided in SEQ ID NOS:6 and 7, additional hTRT intron and flankingsequences may be readily obtained using routine molecular biologicaltechniques. For example, additional hTRT genomic sequence may beobtained from Lambda clone GΦ5 (ATCC Accession No. 209024), describedsupra and in Example 4. Still other hTRT genomic clones and sequencesmay be obtained by screening a human genomic library using an hTRTnucleic acid probe having a sequence or subsequence from SEQ ID NO:1.Additional clones and sequences (e.g., still further upstream) may beobtained by using labeled sequences or subclones derived from λGΦ5 toprobe appropriate libraries. Other useful methods for furthercharacterization of hTRT flanking sequences include those generalmethods described by Gobinda et al., 1993, PCR Meth. Applic. 2:318;Triglia et al., 1988, Nucleic Acids Res. 16:8186; Lagerstrom et al.,1991, PCR Methods Applic. 1:111; and Parker et al., 1991, Nucleic AcidsRes. 19:3055.

[0179] Intronic sequences can be identified by routine means such as bycomparing the hTRT genomic sequence with hTRT cDNA sequences (see, e.g.,Example 3), by S1 analysis (see Ausubel et al., supra, at Chapter 4), orvarious other means known in the art. Intronic sequences can also befound in pre-mRNA (i.e., unspliced or incompletely spliced mRNAprecursors), which may be amplified or cloned following reversetranscription of cellular RNA.

[0180] When desired, the sequence of the cloned, amplified, or otherwisesynthesized hTRT or other TRT nucleic acid can be determined or verifiedusing DNA sequencing methods well known in the art (see, e.g., Ausubelet al., supra). Useful methods of sequencing employ such enzymes as theKlenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp,Cleveland Ohio), Taq DNA polymerase (Perkin Elmer, Norwalk Conn.),thermostable T7 polymerase (Amersham, Chicago Ill.), or combinations ofrecombinant polymerases and proofreading exonucleases such as theELONGASE Amplification System marketed by Gibco BRL (Gaithersburg Md.).When sequencing or verifying the sequence of oligonucleotides (such asoligonucleotide made de novo by chemical synthesis), the method of Maxamand Gilbert may be preferred (Maxam and Gilbert, 1980, Meth. Enz.65:499; Ausubel et al., supra, Ch. 7).

[0181] The 5′ untranslated sequences of hTRT or other TRT mRNAs can bedetermined directly by cloning a “full-length” hTRT or other cDNA usingstandard methods such as reverse transcription of mRNA, followed bycloning and sequencing the resulting cDNA. Preferred oligo(dT)-primedlibraries for screening or amplifying full length cDNAs that have beensize-selected to include larger cDNAs may be preferred. Random primedlibraries are also suitable and often include a larger proportion ofclones that contain the 5′ regions of genes. Other well known methodsfor obtaining 5′ RNA sequences, such as the RACE protocol described byFrohman et al., 1988, Proc. Nat. Acad. Sci USA 85:8998, may also beused. If desired, the transcription start site of an hTRT or other TRTmRNA can be determined by routine methods using the nucleic acidsprovided herein (e.g., having a sequence of SEQ ID NO:1). One method isS1 nuclease analysis (Ausubel et al., supra) using a labeled DNA havinga sequence from the 5′ region of SEQ ID NO:1.

[0182] 2) Chemical Synthesis of Nucleic Acids

[0183] The present invention also provides hTRT polynucleotides (RNA,DNA or modified) that are produced by direct chemical synthesis.Chemical synthesis is generally preferred for the production ofoligonucleotides or for oligonucleotides and polynucleotides containingnonstandard nucleotides (e.g., probes, primers and antisenseoligonucleotides). Direct chemical synthesis of nucleic acids can beaccomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109 (1979); thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859(1981); and the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis typically produces a single stranded oligonucleotide,which may be converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase andan oligonucleotide primer using the single strand as a template. One ofskill will recognize that while chemical synthesis of DNA is oftenlimited to sequences of about 100 or 150 bases, longer sequences may beobtained by the ligation of shorter sequences or by more elaboratesynthetic methods.

[0184] It will be appreciated that the hTRT (or hTR or other)polynucleotides and oligonucleotides of the invention can be made usingnonstandard bases (e.g., other than adenine, cytidine, guanine, thymine,and uridine) or nonstandard backbone structures to provides desirableproperties (e.g., increased nuclease-resistance, tighter-binding,stability or a desired T_(M)). Techniques for rendering oligonucleotidesnuclease-resistant include those described in PCT publication WO94/12633. A wide variety of useful modified oligonucleotides may beproduced, including oligonucleotides having a peptide-nucleic acid (PNA)backbone (Nielsen et al., 1991, Science 254:1497) or incorporating2′-O-methyl ribonucleotides, phosphorothioate nucleotides, methylphosphonate nucleotides, phosphotriester nucleotides, phosphorothioatenucleotides, phosphoramidates. Still other useful oligonucleotides maycontain alkyl and halogen-substituted sugar moieties comprising one ofthe following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃,OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n is from 1 toabout 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a cholesteryl group; a folate group; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. Folate, cholesterol or other groups whichfacilitate oligonucleotide uptake, such as lipid analogs, may beconjugated directly or via a linker at the 2′ position of any nucleosideor at the 3′ or 5′ position of the 3′-terminal or 5′-terminalnucleoside, respectively. One or more such conjugates may be used.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group. Other embodiments may include atleast one modified base form or “universal base” such as inosine, orinclusion of other nonstandard bases such as queosine and wybutosine aswell as acetyl-, methyl-, thio- and similarly modified forms of adenine,cytidine, guanine, thymine, and uridine which are not as easilyrecognized by endogenous endonucleases. The invention further providesoligonucleotides having backbone analogues such as phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene (methylimino), 3′-N-carbamate, morpholino carbamate,chiral-methyl phosphonates, nucleotides with short chain alkyl orcycloalkyl intersugar linkages, short chain heteroatomic or heterocyclicintersugar (“backbone”) linkages, or CH₂—NH—O—CH₂, CH₂—N(CH₃)—OCH₂,CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones(where phosphodiester is O—P—O—CH₂), or mixtures of the same. Alsouseful are oligonucleotides having morpholino backbone structures (U.S.Pat. No. 5,034,506).

[0185] Useful references include Oligonucleotides and Analogues, APractical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992);Milligan et al., 9 Jul. 1993, J. Med. Chem. 36(14):1923-1937; AntisenseResearch and Applications (1993, CRC Press), in its entirety andspecifically Chapter 15, by Sanghvi, entitled “Heterocyclic basemodifications in nucleic acids and their applications in antisenseoligonucleotides.” Antisense Therapeutics, ed. Sudhir Agrawal (HumanaPress, Totowa, N.J., 1996).

[0186] D) Labeling Nucleic Acids

[0187] It is often useful to label the nucleic acids of the invention,for example, when the hTRT or other oligonucleotides or polynucleotidesare to be used as nucleic acid probes. The labels (see infra) may beincorporated by any of a number of means well known to those of skill inthe art. In one embodiment, an unamplified nucleic acid (e.g., mRNA,polyA mRNA, cDNA) is labeled. Means of producing labeled nucleic acidsare well known to those of skill in the art and include, for example,nick-translation, random primer labeling, end-labeling (e.g. using akinase), and chemical conjugation (e.g., photobiotinylation) orsynthesis. In another embodiment, the label is simultaneouslyincorporated during an amplification step in the preparation of thesample nucleic acids. Thus, for example, polymerase chain reaction (PCR)or other nucleic acid amplification method with labeled primers orlabeled nucleotides will provide a labeled amplification product. Inanother embodiment, transcription amplification using a labelednucleotide (e.g. fluorescein-labeled UTP and/or CTP) incorporates alabel into the transcribed nucleic acids. An amplification product mayalso, or alternatively, be labeled after the amplification is completed.

[0188] E) Illustrative Oligonucleotides

[0189] As noted supra and discussed in detail infra, oligonucleotidesare used for a variety of uses including as primers, probes, therapeuticor other antisense oligonucleotides, triplex oligonucleotides, andnumerous other uses as apparent from this disclosure. Table 2 providescertain illustrative specific oligonucleotides that may be used in thepractice of the invention. It will be appreciated that numerous otheruseful oligonucleotides of the invention may be synthesized by one ofskill, following the guidance provided herein.

[0190] In Table 2, “seq” means that the primer has been used, or isuseful, for sequencing; “PCR” means that the primer has been used, or isuseful, for PCR; “AS” means that means that the primer has been used, oris useful for antisense inhibition of telomerase activity; “CL” meansthat the primer has been used, or is useful in cloning regions of hTRTgenes or RNA, “mut” means that the primer has been used, or is usefulfor constructing mutants of hTRT genes or gene products. “UC,” means“upper case,” and “lc” means “lower case.” Mismatches and insertions(relative to SEQ ID NO:1) are indicated by underlining; deletions areindicated by a “−”. It will be appreciated that nothing in Table 2 isintended to limit the use of any particular oligonucleotide to anysingle use or set of uses. TABLE 2 USEFUL OLIGONUCLEOTIDES primer5′-sequence-3′* Notes TCP1.1 GTGAAGGCACTGTTCAGCG TCP1.2GTGGATGATTTCTTGTTGG TCP1.4 CTGGACACTCAGCCCTTGG TCP1.5GGCAGGTGTGCTGGACACT TCP1.6 TTTGATGATGCTGGCGATG TCP1.7GGGGCTCGTCTTCTACAGG TCP1.8 CAGCAGGAGGATCTTGTAG TCP1.9TGACCCCAGGAGTGGCACG TCP1.10 TCAAGCTGACTCGACACCG TCP1.11CGGCGTGACAGGGCTGC TCP1.12 GCTGAAGGCTGAGTGTCC TCP1.13 TAGTCCATGTTCACAATCGTCP1.14 CTGTGCTGGGCCTGGACGATA TCP1.15 CGCGTGGGTGAGGTGAGGTG TCP1.16TTTCCGTGTTGAGTGTTTC TCP1.17 GTCACCGTGTTGGGCAGG TCP1.19GCTACCTGCCCAACACGG TCP1.20 GCGCGAAGAACGTGCTGG TCP1.21CA-CTGCTCCTTGTCGCCTG TCP1.22 TTCCCAAGGACTTTGTTGC TCP1.24TGTTCCTCAAGACGCACTG TCP1.25 TACTGCGTGCGTCGGTATG TCP1.26GGTCTTGCGGCTGAAGTGT TCP1.27 TGGTTCACCTGCTGGCACG TCP1.28GTGGTTTCTGTGTGGTGTC TCP1.29 GACACCACACAGAAACCAC TCP1.30GTGCCAGCAGGTGAACCAG TCP1.32B GCAGTGCGTCTTGAGGAGC TCP1.33TGGAACCATAGCGTCAGGGAG TCP1.34 GGCCTCCCTGACGCTATGGTT TCP1.35GC(GT)CGGCGCTGCCACTCAGG TCP1.35t GCTCGGCGCTGCCACTCAGG TCP1.36ACGCCGAGACCAAGCACTTC TCP1.38 CCAAAGAGGTGGCTTCTTCG TCP1.39AAGGCCAGCACGTTCTTCGC TCP1.40 CACGTTCGTGCGGCGCCTG TCP1.41CCTTCACCACCAGCGTGCG TCP1.42 GGCGACGACGTGCTGGTTC TCP1.43GGCTCAGGGGCAGCGCCAC TCP1.44 CTGGCAGGTGTACGGCTTC TCP1.45GCGTGGACCGAGTGACCGTGGTTTC TCP1.46 GACGTGGTGGCCGCGATGTGG TCP1.47GAAGTCTGCCGTTGCCCAAGAG TCP1.48 GACACCACACAGAAACCACGGTCAC TCP1.49CGCCCCCTCCTTCCGCCAGGT TCP1.50 CGAAGCCGAAGGCCAGCACGTTCTT TCP1.51GGTGGCCCGAGTGCTGCAGAGG TCP1.52 GTAGCTGCGCACGCTGGTGGTGAAG TCP1.53TGGGCGACGACGTGCTGGTTCA TCP1.54 TATGGTTCCAGGCCCGTTCGCATCC TCP1.55CCAGCTGCGCCTACCAGGTGTGC TCP1.56 GGCCTCCCTGACGCTATGGTTCCAG TCP1.57GGTGCTGCCGCTGGCCACGTTCG TCP1.58 TCCCAGGGCACGCACACCAGGCACT TCP1.59GTACAGGGCACACCTTTGGTCACTC TCP1.60 TCGACGACGTACACACTCATCAGCC TCP1.61AGCGGCAGCACCTCGCGGTAGTGGC TCP1.62 CCACCAGCTCCTTCAGGCAGGACAC TCP1.63CCAGGGCTTCCCACGTGCGCAGCAG TCP1.64 CGCACGAACGTGGCCAGCGGCAGCA TCP1.65TGACCGTGGTTTCTGTGTGGTGT TCP1.66 CCCTCTTCAAGTGCTGTCTGATTCC TCP1.67ATCGCGGCCACCACGTCCCT TCP1.68 TGCTCCAGACACTCGGCCGGTAGAA TCP1.69ACGAAGCCCGTACACCTGCC TCP1.72 CGACATCCCTGCGTTCTTGGCTTTC TCP1.73CACTGCTGGCCTCATTCAGGG TCP1.74 GCGACATGGAGAACAAGC TCP1.75GCAGCCATACTCAGGGACAC TCP1.76 CCATCCTCTCCACGCTGCTC TCP1.77GCGATGACCTCCGTGAGCCTG TCP1.78 CCCAGGACAGGCTCACGGA billTCP1CCTCTTCAAGTGCTGTCTGATTCC billTCP2 CAGCTCGACGACGTACACACTCATC billTCP4CTGACGTCCAGACTCCGCTTCAT billTCP6 AGCTTGTTCTCCATGTCGCCGTAG rpprim01GACCTGAGCAGCTCGACGACGTACACAC TCATC Lt1 GTCGTCGAGCTGCTCAGGTC Lt2AGCACGCTGAACAGTGCCTT Lt3 GACCTGAGCAGCTCGACGAC Lt4 AAGGCACTGTTCAGCGTGCTLt5 CGGCCGAGTGTCTGGAGCAA Lt6 GGATGAAGCGGAGTCTGGA BamH1Lt7ATGGATCCGTCGTCGAGCTGCTCAGGTCT BamH1 site Sal1Lt8 ATCAGC Pvu II site (notSal I) TGAGCACGCTGAACAGTGCCTTC K303 GTCTCCGTGACATAAAAGAAAGAC K304GCCAAGTTCCTGCACTGGCT K305 GCCTGTTCTTTTGAAACGTGGTCT K306XXGCCTGTTCTTTTGAAACGTGGTCT X = biotin, = K305 K311GTCAAGATGCCTGAGATAGAAC K312 TGCTTAGCTTGTGGGGGTGTCA K313TGCTTAGCTTGTGGGGGTGTCA K320 GCTGCGTCCTGCTGCGCACGT K321CAGCGGGGAGCGCGCGGCATC K322 TGGGCCACCAGCGCGCGGAAA slanti.1CGGCCGCAGCCCGTCAGGCTTGGGG slanti.2 CCGACAGCTCCCGCAGCTGCACCC slanti.3CGTACACACTCATCAGCCAGTGCAGGAA CTTGGC slanti.4CGCGCCCGCTCGTAGTTTGAGCACGCTGA ACAGTGCCTTC ACCCTCG slanti.5GCGGAGTCTGGACGTCAGCAGGGCGGGC CTGGCTTCCCG UTR2ATTTGACCCACAGGGACCCCCATCCAG FW5 ATGACCGCCCTCCTCGTGAG Nam1GCCACCCCCGCGATGCC Nam2 AGCCCTGGCCCCGGCCA Nam3 TCCCACGTGCGCAGCAG Nam4AGCAGGACGCAGCGCTG PE01 CGCGGTAGTGGCTGCGCAGCAGGGAGCG CACGGC PE02CCAGGGCTTCCCACGTGCGCAGCAGGAC GCAGCGC LM101 CTAGTCTAGATCA/ Xba I site/HAtag/hTRT GCTAGCGTAATCTGGAACATCGTA into pGRN121TGGGTA/GTCCAGGATGGTCTTGAAGTC LM103 TACCATGGGCTACCCATACGACGTTCCAG insertsHA tag into a Nde I site ATTACGCTCA at 5′ end of hTRT LM104TATGAGCGTAATCTGGAACGTCGTATGGG anneals to LM103 TAGCCCATGG LM105GTGTACGTCGTCGAGCTCCTCAGGTCTGC change = F560A CTTTT (phe > ala)ATGTCACGGAG LM106 GTGTACGTCGTCGAGCTCCTCAGGTCCTTT change = F561ACGCTTATGTC (phe > ala) ACGGAGACC LM107 CCTCAGGTCTTTCTTTGCTGTCACGGAGAchange = Y562A CAACGTTT (tyr > ala) CAAAAGAACAG LM108GGTCTTTCTTTTATGTCGCGGAGACAACG change = T564A TTT (thr > ala) CAAAAGAACAGLM109 CTTTCTTTTATGTCACGGCGACAACGTTT change = E565A CAAAAGAACA G LM_FFYTEATGAGTGTGTACGTCGTCGAGCTCCTCAG deletion of FFYVTE GTCTACCACG (aa560-565)TTTCAAAAGAACAGGCTCTTTTC TCP061: GGCTGATGAGTGTGTACGTCGTCGA complement toTCP1.61 HUMO1: ACGTGGTCTCCGTGACATAAAAGAA to DD motif, designed topossibly anneal to mTRT HUMO2: AGGTCTTTCTTTTATGTCACGGA to DD motif,designed to possibly anneal to mTRT HUMO3: CACAGACCCCCGTCGCCTGGTCdesigned to possibly anneal to mTRT HUMO4: CGGAGTCTGGACGTCAGCAGGGCdesigned to possibly anneal to mTRT SLW F1NcgcggatccgtaactaaaATGCCGCGCGCTCCCCG for GST fusion construct (782 to1636) CTGC UC = hTRT seq, 1c = BamH I site + 2 stop codons SLW F1CccggaattcgttagttacttaCAAAGAGGTGGCTTCTT for GST fusion construct (782 to1636) CGGC UC = hTRT seq, 1c = EcoR I site + 3 stop codons SLW F1N/SLWF1C amplify a 893 nt piece of pGRN121 (782 to 1636) SLW F2NcgcggatccgtaactaaaGCCACCTCTTTGGAGGGTGCG for GST fusion construct (1625to 2458) UC = hTRT seq, 1c = BamH1 Site + 2 stop codons SLW F2CccggaattcgttagttacttaAGACCTGAGCAGCTCGACGAC for GST fusion construct(1625 to 2458) UC = hTRT seq, 1c = EcoR I site + 3 stop codons SLWF2N/SLW F2C amplify a 872 nt piece of pGRN121 (1625 to 2458) SLW F3NcgcggatccgtaactaaaATGAGTGTGTACGTCGTCGAG for GST fusion construct (2426to 3274) UC = hTRT seq, 1c = BamH1 site + 2 stop codons SLW F3CccggaattcgttacttacttaGATCCCCTGGCACTGGACG for GST fusion construct (2426to 3274) UC = hTRT seq, 1c = EcoR I site + 3 stop codons SLW F3N/SLW F3Camplify a 887 nt piece of pGRN121 (2426 to 3274) SLW F4NcgcggatccgtaactaaaATCCCGCAGGGCTCCATCCTC for GST fusion construct (3272to 4177) UC = hTRT seq, 1c = BamH1 site + 2 stop codons SLW F4CccggaattcgttagttacttaGTCCAGGATGGTCTTGAAGTC for GST fusion construct(3272 to 4177) UC = hTRT seq, 1c = EcoR I site + 3 stop codons SLWF4N/SLW F4C amplify a 944 nt piece of pGRN121 (3272 to 4177) 40-60GGCATCGCGGGGGTGGCCGGG phosphorothioate 260-280 GGACACCTGGCGGAAGGAGGGphosphorothioate 500-520 GCGTGCCAGCAGGTGAACCAG phosphorothioate 770-790CTCAGGGGCAGCGCCACGCCT phosphorothioate 885-905 AGGTGGCTTCTTCGGCGGGTCphosphorothioate 1000-1020 GGACAAGGCGTGTCCCAGGGA phosphorothioate1300-1320 GCTGGGGTGACCGCAGCTCGC phosphorothioate 1520-1540GATGAACTTCTTGGTGTTCCT phosphorothioate 2110-2130 GTGCGCCAGGCCCTGTGGATAphosphorothioate 2295-2315 GCCCATGGGCGGCCTTCTGGA phosphorothioate2450-2470 GAGGCCACTGCTGGCCTCATT phosphorothioate 2670-2690GGGTGAGGTGAGGTGTCACCA phosphorothioate 3080-3110GCTGCAGCACACATGCGTGAAACCTGTACGC phosphorothioate 3140-3160GACGCGCAGGAAAAATGTGGG phosphorothioate 3690-3710 CCGAGCGCCAGCCTGTGGGGAphosphorothioate 55-75 CAGCGGGGAGCGCGCGGCATC phosphorothioate 151-171CAGCACCTCGCGGTAGTGGCT phosphorothioate TP1.1 TCAAGCCAAACCTGAATCTGAGTP1.2 CCCGAGTGAATCTTTCTACGC TP1.3 GTCTCTGGCAGTTTCCTCATCCC TP1.4TTTAGGCATCCTCCCAAGCACA USE SEQ primer mismatch?* seq PCR AS CL MUT IDTCP1.1 x x 377 TCP1.2 x x 381 TCP1.4 x x 382 TCP1.5 x x 383 TCP1.6 x x384 TCP1.7 Y x x 385 TCP1.8 x x 386 TCP1.9 x x 387 TCP1.10 x x 388TCP1.11 x x 389 TCP1.12 x x 390 TCP1.13 x x 391 TCP1.14 x x 379 TCP1.15x x 378 TCP1.16 x x 392 TCP1.17 x x 393 TCP1.19 x x 394 TCP1.20 x x 395TCP1.21 Y x x 396 TCP1.22 x x 397 TCP1.24 Y x x 398 TCP1.25 x x 399TCP1.26 x x 400 TCP1.27 x x 401 TCP1.28 x x 402 TCP1.29 x x 403 TCP1.30x x 404 TCP1.32B x x 405 TCP1.33 x x 406 TCP1.34 x x 407 TCP1.35 x x 408TCP1.35t 409 TCP1.36 x x 410 TCP1.38 x x 411 TCP1.39 x x 412 TCP1.40 x x413 TCP1.41 x x 414 TCP1.42 x x 415 TCP1.43 x x 416 TCP1.44 x x 417TCP1.45 x x 418 TCP1.46 x x 419 TCP1.47 x x 420 TCP1.48 x x 421 TCP1.49x x 422 TCP1.50 x x 423 TCP1.51 x x 424 TCP1.52 x x 425 TCP1.53 x x 426TCP1.54 x x 427 TCP1.55 x x 428 TCP1.56 x x 429 TCP1.57 x x 430 TCP1.58x x 431 TCP1.59 x x 432 TCP1.60 x x 433 TCP1.61 x x 434 TCP1.62 x x 435TCP1.63 x x 436 TCP1.64 x x 437 TCP1.65 x x 438 TCP1.66 x x 439 TCP1.67x x 440 TCP1.68 x x 441 TCP1.69 x x 442 TCP1.72 x x 443 TCP1.73 x x 444TCP1.74 x x 445 TCP1.75 x x 446 TCP1.76 x x 447 TCP1.77 x x 448 TCP1.78x x 449 billTCP1 x x 450 billTCP2 x x 451 billTCP4 x x 452 billTCP6 x x380 rpprim01 x x 453 Lt1 x x 454 Lt2 x x 455 Lt3 x x 456 Lt4 x x 457 Lt5Y x x 458 Lt6 x x 459 BamH1Lt7 Y x x 460 Sal1Lt8 Y x x 461 K303 x x 462K304 x x 463 K305 x x 464 K306 x x 465 K311 x x 466 K312 x x 467 K313 xx 467 K320 x x 468 K321 x x 469 K322 x x 470 slanti.1 Y x x 471 slanti.2Y x x 472 slanti.3 x x 473 slanti.4 x x 474 slanti.5 x x 475 UTR2 x x476 FW5 x x 477 Nam1 x x 478 Nam2 x x 479 Nam3 x x 480 Nam4 x x 481 PE01x x 482 PE02 x x 483 LM101 x 484 LM103 x 485 LM104 x 486 LM105 x 487LM106 x 488 LM107 x 489 LM108 x 490 LM109 x 491 LM_FFYTE x 492 TCP061: xx 493 HUMO1: x x x 494 HUMO2: x x x 495 HUMO3: x x x 496 HUMO4: x x x497 SLW F1N x x 498 SLW F1C x x 499 SLW F2N x x 500 SLW F2C x x 501 SLWF3N x x 502 SLW F3C x x 503 SLW F4N x x 504 SLW F4C x x 505 40-60 x 506260-280 x 507 500-520 x 508 770-790 x 509 885-905 x 510 1000-1020 x 5111300-1320 x 512 1520-1540 x 513 2110-2130 x 514 2295-2315 x 5152450-2470 x 516 2670-2690 x 517 3080-3110 x 518 3140-3160 x 5193690-3710 x 520 55-75 x 521 151-171 x 522 TP1.1 x 523 TP1.2 x 524 TP1.3x 525 TP1.4 x 526

[0191] IV. TRT Proteins and Peptides

[0192] A) Generally

[0193] The invention provides a wide variety of hTRT proteins usefulfor, inter alia, production of telomerase activity, inhibition oftelomerase activity in a cell, induction of an anti-hTRT immuneresponse, as a therapeutic reagent, as a standard or control in adiagnostic assay, as a target in a screen for compounds capable ofactivation or inhibition of an activity of hTRT or telomerase, andnumerous other uses that will be apparent to one of skill or areotherwise described herein. The hTRT of the invention includefunctionally active proteins (useful for e.g., conferring telomeraseactivity in a telomerase-negative cell) and variants, inactive variants(useful for e.g., inhibiting telomerase activity in a cell), hTRTpolypeptides, and telomerase RNPs (e.g., ribonucleoprotein complexescomprising the proteins) that exhibit one, several, or all of thefunctional activities of naturally occurring hTRT and telomerase, asdiscussed in greater detail for illustrative purposes, below.

[0194] In one embodiment, the hTRT protein of the invention is apolypeptide having a sequence as set forth in FIG. 17 (SEQ ID NO:2), ora fragment thereof. In another embodiment, the hTRT polypeptide differsfrom SEQ ID NO:2 by internal deletions, insertions, or conservativesubstitutions of amino acid residues. In a related embodiment, theinvention provides hTRT polypeptides with substantial similarity to SEQID NO:2. The invention further provides hTRT polypeptides that aremodified, relative to the amino acid sequence of SEQ ID NO:2, in somemanner, e.g., truncated, mutated, derivatized, or fused to othersequences (e.g., to form a fusion protein). Moreover, the presentinvention provides telomerase RNPs comprising an hTRT protein of theinvention complexed with a template RNA (e.g., hTR). In otherembodiments, one or more telomerase-associated proteins is associatedwith hTRT protein and/or hTR.

[0195] The invention also provides other naturally occurring hTRTspecies or normaturally occurring variants, such as proteins having thesequence of, or substantial similarity to SEQ ID NO:5 [[FIG. 20], SEQ IDNO:10 [FIG. 19], and fragments, variants, or derivatives thereof.

[0196] The invention provides still other hTRT species and variants. Oneexample of an hTRT variant may result from ribosome frameshifting ofmRNA encoded by the clone 712562 (SEQ ID NO:3 [FIG. 18]) or the pro90variant hTRT shown in SEQ ID NO:4 [FIG. 20] and so result in thesynthesis of hTRT polypeptides containing all the TRT motifs (for ageneral example, see, e.g., Tsuchihashi et al., 1990, Proc. Natl. Acad.Sci. USA 87:2516; Craigengen et al., 1987, Cell 50: 1; Weiss, 1990, Cell62:117). Ribosome frameshifting can occur when specific mRNA sequencesor secondary structures cause the ribosome to “stall” and jump onenucleotide forwards or back in the sequence. Thus, a ribosome frameshiftevent on the 712562 mRNA could cause the synthesis of an approximately523 amino acid residue polypeptide. A ribosome frameshift event on thepro90 sequence could result in a protein with approximately 1071residues. It will be appreciated that proteins resulting from ribosomeframeshifting can also be expressed by synthetic or recombinanttechniques provided by the invention.

[0197] Human TRT proteins, peptides, and functionally equivalentproteins may be obtained by purification, chemical synthesis, orrecombinant production, as discussed in greater detail below.

[0198] B) TRT Protein Activities

[0199] The TRT polypeptides of the invention (including fragments,variants, products of alternative alleles, and fusion proteins) can haveone or more, or all of the functional activities associated with nativehTRT. Except as noted, as used herein, an hTRT or other TRT polypeptideis considered to have a specified activity if the activity is exhibitedby either the hTRT protein without an associated RNA (e.g., hTR) or inan hTRT-associated RNA (e.g., hTR) complex. The hTR-binding activity ofhTRT is one example of an activity associated with the hTRT protein.Methods for producing complexes of nucleic acids (e.g., hTR) and thehTRT polypeptides of the invention are described infra.

[0200] Modification of the hTRT protein (e.g., by chemical orrecombinant means, including mutation or modification of apolynucleotide encoding the hTRT polypeptide or chemical synthesis of apolynucleotide that has a sequence different than a nativepolynucleotide sequence) to have a different complement of activitiesthan native hTRT can be useful in therapeutic applications or inscreening for specific modulators of hTRT or telomerase activity. Inaddition, assays for various hTRT activities can be particularly usefulfor identification of agents (e.g., activity modulating agents) thatinteract with hTRT or telomerase to change telomerase activity.

[0201] The activities of native hTRT, as discussed infra, includetelomerase catalytic activity (which may be either processive ornon-processive activity); telomerase processivity; conventional reversetranscriptase activity; nucleolytic activity; primer or substrate(telomere or synthetic telomerase substrate or primer) binding activity;dNTP binding activity; RNA (i.e., hTR) binding activity; and proteinbinding activity (e.g., binding to telomerase-associated proteins,telomere-binding proteins, or to a protein-telomeric DNA complex). Itwill be understood, however, that present invention also provides hTRTcompositions without any particular hTRT activity but with some usefulactivity related to the hTRT or other TRT proteins (e.g., certaintypically short immunogenic peptides, inhibitory peptides).

[0202] 1) Telomerase Catalytic Activity

[0203] As used herein, a polypeptide of the invention has “telomerasecatalytic activity,” when the polypeptide is capable of extending a DNAprimer that functions as a telomerase substrate by adding a partial,one, or more than one repeat of a sequence (e.g., TTAGGG) encoded by atemplate nucleic acid (e.g., hTR). This activity may be processive ornonprocessive. Processive activity occurs when a telomerase RNP addsmultiple repeats to a primer or telomerase before the DNA is released bythe enzyme complex. Non-processive activity occurs when telomerase addsa partial, or only one, repeat to a primer and is then released. Invivo, however, a non-processive reaction could add multiple repeats bysuccessive rounds of association, extension, and dissociation. This canoccur in vitro as well, but it is not typically observed in standardassays due to the vastly large molar excess of primer over telomerase instandard assay conditions.

[0204] To characterize an hTRT polypeptide as having non-processiveactivity, a conventional telomerase reaction is performed usingconditions that favor a non-processive reaction, for example hightemperatures (i.e., 35-40EC, typically 37EC), low dGTP concentrations (1μM or less), high primer concentrations (5 μM or higher), and highdATP/TTP concentrations (2 mM or higher), with the temperature and dGTPtypically having the greatest effect. To characterize an hTRTpolypeptide as having processive activity, a conventional telomerasereaction is performed using conditions that favor a processive reaction(for example, 27-34° C., typically 30° C.), high dGTP concentration (10μM or higher), low primer concentration (1 μM or lower), and/or low dATPand TTP concentrations (0.3-1 mM) with temperature and dGTP typicallyconcentration being the most critical. Alternatively, a TRAP assay (forprocessive or moderately processive activity) or the dot-blot and gelblot assays (for processive activity) may be used. The hTRT polypeptideof the invention can possess a non-processive activity, but not aprocessive activity (e.g., if an alteration of the hTRT polypeptidereduces or eliminates the ability to translocate), can be solelyprocessive, or can possess both activities.

[0205] a) Non-Processive Activity

[0206] A non-processive telomerase catalytic activity can extend the DNAprimer from the position where the 3′ end anneals to the RNA template tothe 5′ end of the template sequence, typically terminating with theaddition of the first G residue (as, for example, when the template ishTR). As shown below, the exact number of nucleotides added is dependenton the position of the 3′ terminal nucleotide of the primer in theTTAGGG repeat sequence. NONPROCESSIVE ACTIVITY i) ---------TTAGGGttag(DNA) SEQ ID NO:527    3′-----AUCCCAAUC-----5′ (RNA) ii)---------TTAGggttag (DNA) SEQ ID NO:527    3′-----AUCCCAAUC-----5′ (RNA)

[0207] Thus, 4 nucleotides are added to the --TTAGGG primer (i) while 6nucleotides are added to the --TTAG primer (ii). The first repeat addedby telomerase in a processive reaction is equivalent to this step;however, in a processive reaction telomerase performs a translocationstep where the 3′ end is released and re-bound at the 3′ region of thetemplate in a position sufficient to prime addition of another repeat(see Morin, 1997, Eur. J. Cancer 33:750).

[0208] A fully non-processive reaction produces only one band in aconventional assay using a single synthetic primer. Because this resultcould also be produced by other enzymes, such as a terminal transferaseactivity, it may be desirable in some applications to verify that theproduct is a result of a telomerase catalytic activity. A telomerase(comprising hTRT) generated band can be distinguished by severaladditional characteristics. The number of nucleotides added to the endof the primer should be consistent with the position of the primer 3′end. Thus, a --TTAGGG primer should have 4 nucleotides added and a--TTAG primer should have 6 nucleotides added (see above). In practice,two or more sequence permuted primers can be used which have the sameoverall length but different 5′ and 3′ endpoints. As an illustrativeexample, the non-processive extension of primers 5′-TTAGGGTTAGGGTTAGGG(SEQ ID NO:528) and 5′-GTTAGGGTTAGGGTTAGG (SEQ ID NO:529) will generateproducts whose absolute length will be one nucleotide different (4 addedto 5′-TTAGGGTTAGGGTTAGGG (SEQ ID NO:528) for a 22 nt total length, and 5added to 5′-GTTAGGGTTAGGGTTAGG (SEQ ID NO:529) for a 23 nt totallength). The nucleotide dependence of the reaction should be consistentwith the position of the primer terminus. Thus, a --TTAGGG primerproduct should require dGTP, TTP, and dATP, but not dCTP, and a---AGGGTT primer product should require dGTP and dATP, but not TTP ordCTP. The activity should be sensitive to RNAase or micrococcal nucleasepre-treatment (see Morin, 1989, Cell 59: 521) under conditions that willdegrade hTR and so eliminate the template.

[0209] b) Processive Activity

[0210] In practice, a processive activity is easily observed by theappearance of a six nucleotide ladder in a conventional assay, TRAPassay, or gel-blot assay. A dot-blot assay can also be used, but noladder is detected in such a method. The conventional assay is describedin Morin, 1989, Cell 59:521, which is incorporated herein in itsentirety and for all purposes. The TRAP assay is described in U.S. Pat.No. 5,629,154; see also, PCT publication WO 97/15687, PCT publication WO95/13381; Krupp et al. Nucleic Acids Res., 1997, 25: 919; and Wright etal., 1995, Nuc. Acids Res. 23:3794, each of which is incorporated hereinin its entirety and for all purposes. The dot blot immunoassay isdescribed in detail in co-pending U.S. patent application Ser. No.08/833,377, filed Apr. 14, 1997, which is incorporated herein byreference in its entirety and for all purposes. The dot blot assay canbe used in a format in which a non-processive activity, which does notadd the 3 or more repeats required for stable hybridization of the(CCCUAA)n probe used to detect the activity, is tested with compounds orhTRT variants to determine if the same generates processivity, i.e., ifthe probe detects an expected telomerase substrate, then the compound ormutant is able to change the non-processive activity to a processiveactivity. Other assays for processive telomerase catalytic activity canalso be used, e.g., the stretch PCR assay of Tatematsu et al., 1996,Oncogene 13:2265. The gel-blot assay, a combination of the conventionaland dot blot assays can also be used. In this variation a conventionalassay is performed with no radiolabeled nucleotide and with high dGTPconcentrations (e.g., 0.1-2 mM). After performing the conventionalassay, the synthesized DNA is separated by denaturing PAGE andtransferred to a membrane (e.g., nitrocellulose). Telomeric DNA (theproduct of telomerase—an extended telomerase primer or substrate) canthen be detected by methods such as hybridization using labeledtelomeric DNA probes (e.g., probes containing the CCCTAA sequence, asused in the dot blot assay, supra). An advantage of this technique isthat it is more sensitive than the conventional assay and providesinformation about the size of the synthesized fragments and processivityof the reaction.

[0211] c) Activity Determinations

[0212] The telomerase activity of an hTRT polypeptide can be determinedusing an unpurified, partially purified or substantially purified hTRTpolypeptide (e.g., in association with hTR), in vitro, or afterexpression in vivo. For example, telomerase activity in a cell (e.g., acell expressing a recombinant hTRT polypeptide of the invention) can beassayed by detecting an increase or decrease in the length of telomeres.Typically assays for telomerase catalytic activity are carried out usingan hTRT complexed with hTR; however, alternative telomerase templateRNAs may be substituted, or one may conduct assays to measure anotheractivity, such as telomerase-primer binding. Assays to determine thelength of telomeres are known in the art and include hybridization ofprobes to telomeric DNA (an amplification step can be included) and TRFanalysis i.e., the analysis of telomeric DNA restriction fragments[TRFs] following restriction endonuclease digestion, see PCTpublications WO 93/23572 and WO 96/41016; Counter et al., 1992, EMBO J.11:1921; Allsopp et al., 1992, Proc. Nat'l. Acad. Sci. USA 89:10114;Sanno, 1996, Am J Clin Pathol 106:16 and Sanno, 1997, Neuroendocrinology65:299.

[0213] The telomerase catalytic activity of an hTRT polypeptide may bedetermined in a number of ways using the assays supra and othertelomerase catalytic activity assays. According to one method, the hTRTprotein is expressed (e.g., as described infra) in a telomerase negativehuman cell in which hTR is expressed (i.e., either normally in the cellor through recombinant expression), and the presence or absence oftelomerase activity in the cell or cell lysate is determined. Examplesof suitable telomerase-negative cells are IMR 90 (ATCC, #CCL-186) or BJcells (human foreskin fibroblast line; see, e.g., Feng et al., 1995,Science 269:1236). Other examples include retinal pigmented epithelialcells (RPE), human umbilical vein endothelial cells (HUVEC; ATCC#CRL-1730), human aortic endothelial cells (HAEC; Clonetics Corp,#CC-2535), and human mammary epithelial cells (HME; Hammond et al.,1984, Proc. Nat'l. Acad. Sci. USA 81:5435; Stampfer, 1985, J. TissueCulture Methods 9:107). In an alternative embodiment, the hTRTpolypeptide is expressed (e.g., by transfection with an hTRT expressionvector) in a telomerase positive cell, and an increase in telomeraseactivity in the cell compared to an untransfected control cell isdetected if the polypeptide has telomerase catalytic activity. Usuallythe telomerase catalytic activity in a cell transfected with a suitableexpression vector expressing hTRT will be significantly increased, suchas at least about 2-fold, at least about 5-fold, or even at least about10-fold to 100-fold or even 1000-fold higher than in untransfected(control) cells.

[0214] In an alternative embodiment, the hTRT protein is expressed in acell (e.g., a telomerase negative cell in which hTR is expressed) as afusion protein (see infra) having a label or an “epitope tag” to aid inpurification. In one embodiment, the RNP is recovered from the cellusing an antibody that specifically recognizes the tag. Preferred tagsare typically short or small and may include a cleavage site or otherproperty that allows the tag to be removed from the hTRT polypeptide.Examples of suitable tags include the Xpress™ epitope (Invitrogen, Inc.,San Diego Calif.), and other moieties that can be specifically bound byan antibody or nucleic acid or other equivalent method such as thosedescribed in Example 6. Alternative tags include those encoded bysequences inserted, e.g., into SEQ ID NO:1 upstream of the ATG codonthat initiates translation of the protein of SEQ ID NO:2, which mayinclude insertion of a (new) methionine initiation codon into theupstream sequence.

[0215] It will be appreciated that when an hTRT variant is expressed ina cell (e.g., as a fusion protein) and subsequently isolated (e.g., as aribonucleoprotein complex), other cell proteins (i.e.,telomerase-associated proteins) may be associated with (directly orindirectly bound to) the isolated complex. In such cases, it willsometimes be desirable to assay telomerase activity for the complexcontaining hTRT, hTR and the associated proteins.

[0216] 2) Other Telomerase or TRT Protein Activities

[0217] The hTRT polypeptides of the invention include variants that lacktelomerase catalytic activity but retain one or more other activities oftelomerase. These other activities and the methods of the invention formeasuring such activities include (but are not limited to) thosediscussed in the following sections.

[0218] a) Conventional Reverse Transcriptase Activity

[0219] Telomerase conventional reverse transcriptase activity isdescribed in, e.g., Morin, 1997, supra, and Spence et al., 1995, Science267:988. Because hTRT contains conserved amino acid motifs that arerequired for reverse transcriptase catalytic activity, hTRT has theability to transcribe certain exogenous (e.g., non-hTR) RNAs. Aconventional RT assay measures the ability of the enzyme to transcribean RNA template by extending an annealed DNA primer. Reversetranscriptase activity can be measured in numerous ways known in theart, for example, by monitoring the size increase of a labeled nucleicacid primer (e.g., RNA or DNA), or incorporation of a labeled dNTP. See,e.g., Ausubel et al., supra.

[0220] Because hTRT specifically associates with hTR, it can beappreciated that the DNA primer/RNA template for a conventional RT assaycan be modified to have characteristics related to hTR and/or atelomeric DNA primer. For example, the RNA can have the sequence(CCCTAA)_(n), where n is at least 1, or at least 3, or at least 10 ormore (SEQ ID NO:530). In one embodiment, the (CCCTAA)_(n) region is ator near the 5′ terminus of the RNA (similar to the 5′ locations oftemplate regions in telomerase RNAs). Similarly, the DNA primer may havea 3′ terminus that contains portions of the TTAGGG telomere sequence,for example X_(n)TTAG (SEQ ID NO:531), X_(n)AGGG (SEQ ID NO:532),X_(n)(TTAGGG)_(q)TTAG (SEQ ID NOS:533-536), etc., where X is anon-telomeric sequence and n is 8-20, or 6-30, and q is 1-4. In anotherembodiment, the DNA primer has a 5′ terminus that is non-complementaryto the RNA template, such that when the primer is annealed to the RNA,the 5′ terminus of the primer remains unbound. Additional modificationsof standard reverse transcription assays that may be applied to themethods of the invention are known in the art.

[0221] b) Nucleolytic Activity

[0222] Telomerase nucleolytic activity is described in e.g., Morin,1997, supra; Collins and Grieder, 1993, Genes and Development 7:1364.Telomerase possesses a nucleolytic activity (Joyce and Steitz, 1987,Trends Biochem. Sci. 12:288); however, telomerase activity has definingcharacteristics. Telomerase preferentially removes nucleotides, usuallyonly one, from the 3′ end of an oligonucleotide when the 3′ end of theDNA is positioned at the 5′ boundary of the DNA template sequence, inhumans and Tetrahymena, this nucleotide is the first G of the telomericrepeat (TTAGG in humans). Telomerase preferentially removes G residuesbut has nucleolytic activity against other nucleotides. This activitycan be monitored. Two different methods are described here forillustrative purposes. One method involves a conventional telomerasereaction with a primer that binds the entire template sequence (i.e.,terminating at the template boundary; 5′-TAGGGATTAG (SEQ ID NO:537) inhumans). Nucleolytic activity is observed by monitoring the replacementof the last dG residue with a radiolabeled dGTP provided in the assay.The replacement is monitored by the appearance of a band at the size ofthe starting primer as shown by gel electrophoresis and autoradiography.

[0223] A preferred method uses a DNA primer that has a “blocked” 3′terminus that cannot be extended by telomerase. The 3′-blocked primercan be used in a standard telomerase assay but will not be extendedunless the 3′ nucleotide is removed by the nucleolytic activity oftelomerase. The advantage of this method is that telomerase activity canbe monitored by any of several standard means, and the signal is strongand easy to quantify. The blocking of the 3′ terminus of the primer canbe accomplished in several ways. One method is the addition of a3′-deoxy-dNTP residue at the 3′ terminus of the primer using standardoligonucleotide synthesis techniques. This terminus has a 2′ OH but notthe 3′ OH required for telomerase. Other means of blocking the 3′terminus exist, for instance, a 3′ dideoxy terminus, a 3′-amineterminus, and others. An example of a primer for an hTRT nucleolyticassay is 5′-TTAGGGTTAGGGTTA (G_(3′H)) (SEQ ID NO:538) where the lastresidue denotes a 3′-deoxy-guanosine residue (Glen Research, Sterling,Va.). Numerous other variations for a suitable primer based on thedisclosure are known to those of skill in the art.

[0224] c) Primer (Telomere) Binding Activity

[0225] Telomerase primer (telomere) binding activity is described ine.g., Morin, 1997, supra; Collins et al., 1995, Cell 81:677; Harringtonet al, 1995, J. Biol. Chem. 270:8893. Telomerase is believed to have twosites which bind a telomeric DNA primer. The RT motifs associated withprimer binding indicate hTRT and/or hTRT/hTR possesses DNA primerbinding activity. There are several ways of assaying primer bindingactivity; however, a step common to most methods is incubation of alabeled DNA primer with hTRT or hTRT/hTR or other TRT/TR combinationsunder appropriate binding conditions. Also, most methods employ a meansof separating unbound DNA from protein-bound DNA; those methods includethe following.

[0226] i) Gel-shift assays (also called electrophoretic/mobility shiftassays) are those in which unbound DNA primer is separated fromprotein-bound DNA primer by electrophoresis on a nondenaturing gel(Ausubel et al., supra).

[0227] ii) Matrix binding assays include several variations to the basictechnique, which involves binding the hTRT or hTRT/hTR complex to amatrix (e.g., nitrocellulose), either before or after incubation withthe labeled primer. By binding the hTRT to a matrix, the unbound primercan be mechanically separated from bound primer. Residual unbound DNAcan be removed by washing the membrane prior to quantitation. Those ofskill recognize there are several means of coupling proteins to suchmatrices, solid supports, and membranes, including chemical,photochemical, UV cross-linking, antibody/epitope, and non-covalent(hydrophobic, electrostatic, etc.) interactions.

[0228] The DNA primer can be any DNA with an affinity for telomerase,such as, for example, a telomeric DNA primer like (TTAGGG)_(n), where ncould be 1-10 and is typically 3-5 (SEQ ID NO:539). The 3′ and 5′termini can end in any location of the repeat sequence. The primer canalso have 5′ or 3′ extensions of non-telomeric DNA that could facilitatelabeling or detection. The primer can also be derivatized, e.g., tofacilitate detection or isolation.

[0229] d) dNTP Binding Activity

[0230] Telomerase dNTP binding activity is described in e.g., Morin,1997, supra; Spence et al., supra. Telomerase requires dNTPs tosynthesize DNA. The hTRT protein has a nucleotide binding activity andcan be assayed for dNTP binding in a manner similar to other nucleotidebinding proteins (Kantrowitz et al., 1980, Trends Biochem. Sci. 5:124).Typically, binding of a labeled dNTP or dNTP analog can be monitored asis known in the art for non-telomerase RT proteins.

[0231] e) RNA (i.e., hTR) Binding Activity

[0232] Telomerase RNA (i.e., hTR) binding activity is described in e.g.,Morin, 1997, supra; Harrington et al., 1997, Science 275:973; Collins etal., 1995, Cell 81:677. The RNA binding activity of a TRT protein of theinvention may be assayed in a manner similar to the DNA primer bindingassay described supra, using a labeled RNA probe. Methods for separatingbound and unbound RNA and for detecting RNA are well known in the artand can be applied to the activity assays of the invention in a mannersimilar to that described for the DNA primer binding assay. The RNA canbe full length hTR, fragments of hTR or other RNAs demonstrated to havean affinity for telomerase or hTRT. See U.S. Pat. No. 5,583,016 and PCTPub. No. 96/40868.

[0233] 3) Telomerase Motifs as Targets

[0234] The present invention, as noted supra, provides in addition torecombinant hTRT with a full complement (as described supra) ofactivities, hTRT polypeptides having less than the full complement ofthe telomerase activities of naturally occurring telomerase or hTRT orother TRT proteins. It will be appreciated that, in view of thedisclosure herein of the RT and telomerase-specific motifs of TRT,alteration or mutation of conserved amino acid residues, such as arefound in the motif sequences discussed supra, will result in loss-ofactivity mutants useful for therapeutic, drug screening andcharacterization, and other uses. For example, as described in Example1, deletion of motifs B through D in the RT domains of the endogenousTRT gene in S. pombe resulted in haploid cells in which telomereprogressively shortened to the point where hybridization of a telomereprobe to telomeric repeats became almost undetectable, indicating a lossof telomerase catalytic activity. Similarly, alterations in the WxGxS(SEQ ID NO:540) site of motif E can affect telomerase DNA primer bindingor function. Additionally, alterations of the amino acids in the motifsA, B′, and C can affect the catalytic activity of telomerase. Mutationof the DD motif of hTRT can significantly reduce or abolish telomeraseactivity (see Example 16).

[0235] C) Synthesis of hTRT and Other TRT Polypeptides

[0236] The invention provides a variety of methods for making the hTRTand other TRT polypeptides disclosed herein. In the following sections,chemical synthesis and recombinant expression of hTRT proteins,including fusion proteins, is described in some detail.

[0237] 1) Chemical Synthesis

[0238] The invention provides hTRT polypeptides synthesized, entirely orin part, using general chemical methods well known in the art (see e.g.,Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223; and Hornet al., 1980, Nucleic Acids Res. Symp. Ser., 225-232). For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, et al., 1995, Science 269:202), including automated synthesis(e.g., using the Perkin Elmer ABI 431A Peptide Synthesizer in accordancewith the instructions provided by the manufacturer). When full lengthprotein is desired, shorter polypeptides may be fused by condensation ofthe amino terminus of one molecule with the carboxyl terminus of theother molecule to form a peptide bond.

[0239] The newly synthesized peptide can be substantially purified, forexample, by preparative high performance liquid chromatography (e.g.,Creighton, PROTEINS, STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman andCo, New York N.Y. [1983]). The composition of the synthetic peptides (orany other peptides or polypeptides of the invention) may be confirmed byamino acid analysis or sequencing (e.g., the Edman degradationprocedure; Creighton, supra). Importantly, the amino acid sequence ofhTRT, or any part thereof, may be altered during direct synthesis and/orcombined using chemical methods with sequences from other proteins orotherwise, or any part thereof or for any purpose, to produce a variantpolypeptide of the invention.

[0240] 2) Recombinant Expression of hTRT and Other TRT Proteins

[0241] The present invention provides methods, reagents, vectors, andcells useful for expression of hTRT polypeptides and nucleic acids usingin vitro (cell-free), ex vivo or in vivo (cell or organism-based)recombinant expression systems. In one embodiment, expression of thehTRT protein, or fragment thereof, comprises inserting the codingsequence into an appropriate expression vector (i.e., a vector thatcontains the necessary elements for the transcription and translation ofthe inserted coding sequence required for the expression systememployed). Thus, in one aspect, the invention provides for apolynucleotide substantially identical in sequence to an hTRT genecoding sequence at least 25 nucleotides, and preferably for manyapplications 50 to 100 nucleotides or more, of the hTRT cDNAs or genesof the invention, which is operably linked to a promoter to form atranscription unit capable of expressing an hTRT polypeptide. Methodswell known to those skilled in the art can be used to construct theexpression vectors containing an hTRT sequence and appropriatetranscriptional or translational controls provided by the presentinvention (see, e.g., Sambrook et al., supra, Ausubel et al. supra, andthis disclosure).

[0242] The hTRT polypeptides provided by the invention include fusionproteins that contain hTRT polypeptides or fragments of the hTRTprotein. The fusion proteins are typically produced by recombinantmeans, although they may also be made by chemical synthesis. Fusionproteins can be useful in providing enhanced expression of the hTRTpolypeptide constructs, or in producing hTRT polypeptides having otherdesirable properties, for example, comprising a label (such as anenzymatic reporter group), binding group, or antibody epitope. Anexemplary fusion protein, comprising hTRT and enhanced green fluorescentprotein (EGFP) sequences is described in Example 15, infra. It will beapparent to one of skill that the uses and applications discussed inExample 15 and elsewhere herein are not limited to the particular fusionprotein, but are illustrative of the uses of various fusion constructs.

[0243] The fusion protein systems of the invention can also be used tofacilitate efficient production and isolation of hTRT proteins orpeptides. For example, in some embodiments, the non-hTRT sequenceportion of the fusion protein comprises a short peptide that can bespecifically bound to an immobilized molecule such that the fusionprotein can be separated from unbound components (such as unrelatedproteins in a cell lysate). One example is a peptide sequence that isbound by a specific antibody. Another example is a peptide comprisingpolyhistidine tracts e.g. (His)₆ or histidine-tryptophan sequences thatcan be bound by a resin containing nickel or copper ions (i.e.,metal-chelate affinity chromatography). Other examples include Protein Adomains or fragments, which allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). In some embodiments,the fusion protein includes a cleavage site so that the hTRT or otherTRT polypeptide sequence can be easily separated from the non-hTRTpeptide or protein sequence. In this case, cleavage may be chemical(e.g., cyanogen bromide,2-(2-nitrophenylsulphenyl)-3-methyl-3′-bromoindolene, hydroxylamine, orlow pH) or enzymatic (e.g., Factor Xa, enterokinase). The choice of thefusion and cleavage systems may depend, in part, on the portion (i.e.,sequence) of the hTRT polypeptide being expressed. Fusion proteinsgenerally are described in Ausubel et al., supra, Ch. 16, Kroll et al.,1993, DNA Cell. Biol. 12:441, and the Invitrogen 1997 Catalog(Invitrogen Inc, San Diego Calif.). Other exemplary fusion proteins ofthe invention with epitope tags or tags and cleavage sites are providedin Example 6, infra.

[0244] It will be appreciated by those of skill that, although theexpression systems discussed in this section are focused on expressionof hTRT polypeptides, the same or similar cells, vectors and methods maybe used to express hTRT polynucleotides of the invention, includingsense and antisense polynucleotides without necessarily desiringproduction of hTRT polypeptides. Typically, expression of a polypeptiderequires a suitable initiation codon (e.g., methionine), open readingframe, and translational regulatory signals (e.g., a ribosome bindingsite, a termination codon) which may be omitted when translation of anucleic acid sequence to produce a protein is not desired.

[0245] Expression of hTRT polypeptides and polynucleotides may becarried out to accomplish any of several related benefits provided bythe present invention. One illustrative benefit is expression of hTRTpolypeptides that are subsequently isolated from the cell in which theyare expressed (for example for production of large amounts of hTRT foruse as a vaccine or in screening applications to identify compounds thatmodulate telomerase activity). A second illustrative benefit isexpression of hTRT in a cell to change the phenotype of the cell (as ingene therapy applications). Nonmammalian cells can be used forexpression of hTRT for purification, while eukaryotic especiallymammalian cells (e.g., human cells) can be used not only for isolationand purification of hTRT but also for expression of hTRT when a changein phenotype in a cell is desired (e.g., to effect a change inproliferative capacity as in gene therapy applications). By way ofillustration and not limitation, hTRT polypeptides having one or moretelomerase activities (e.g. telomerase catalytic activity) can beexpressed in a host cell to increase the proliferative capacity of acell (e.g., immortalize a cell) and, conversely, hTRT antisensepolynucleotides or inhibitory polypeptides typically can be expressed toreduce the proliferative capacity of a cell (e.g., of a telomerasepositive malignant tumor cell). Numerous specific applications aredescribed herein, e.g., in the discussion of uses of the reagents andmethods of the invention for therapeutic applications, below.

[0246] Illustrative useful expression systems (cells, regulatoryelements, vectors and expression) of the present invention include anumber of cell-free systems such as reticulocyte lysate and wheat germsystems using hTRT polynucleotides in accordance with general methodswell known in the art (see, e.g., Ausubel et al. supra at Ch. 10). Inalternative embodiments, the invention provides reagents and methods forexpressing hTRT in prokaryotic or eukaryotic cells. Thus, the presentinvention provides nucleic acids encoding hTRT polynucleotides,proteins, protein subsequences, or fusion proteins that can be expressedin bacteria, fungi, plant, insect, and animal, including human cellexpression systems known in the art, including isolated cells, celllines, cell cultures, tissues, and whole organisms. As will beunderstood by those of skill, the hTRT polynucleotides introduced into ahost cell or cell free expression system will usually be operably linkedto appropriate expression control sequences for each host or cell freesystem.

[0247] Useful bacterial expression systems include E. coli, bacilli(such as Bacillus subtilus), other enterobacteriaceae (such asSalmonella, Serratia, and various Pseudomonas species) or otherbacterial hosts (e.g., Streptococcus cremoris, Streptococcus lactis,Streptococcus thermophilus, Leuconostoc citrovorum, Leuconostocmesenteroides, Lactobacillus acidophilus, Lactobacillus lactis,Bifidobacterium bifidum, Bifidobacteriu breve, and Bifidobacteriumlongum). The hTRT expression constructs useful in prokaryotes includerecombinant bacteriophage, plasmid or cosmid DNA expression vectors, orthe like, and typically include promoter sequences. Illustrativepromoters include inducible promoters, such as the lac promoter, thehybrid lacZ promoter of the Bluescript7 phagemid [Stratagene, La JollaCalif.] or pSport1 [Gibco BRL]; phage lambda promoter systems; atryptophan (trp) promoter system; and ptrp-lac hybrids and the like.Bacterial expression constructs optionally include a ribosome bindingsite and transcription termination signal regulatory sequences.Illustrative examples of specific vectors useful for expression include,for example, pTrcHis2, (Invitrogen, San Diego Calif.), pThioHis A, B &C, and numerous others known in the art or that may be developed (see,e.g. Ausubel). Useful vectors for bacteria include those that facilitateproduction of hTRT-fusion proteins. Useful vectors for high levelexpression of fusion proteins in bacterial cells include, but are notlimited to, the multifunctional E. coli cloning and expression vectorssuch as Bluescript7 (Stratagene), noted above, in which the sequenceencoding hTRT protein, an hTRT fusion protein or an hTRT fragment may beligated into the vector in-frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced (e.g., pIN vectors; Van Heeke and Schuster, 1989, J.Biol. Chem., 264:5503). Vectors such as pGEX vectors (e.g., pGEX-2TK;Pharmacia Biotech) may also be used to express foreign polypeptides,such as hTRT protein, as fusion proteins with glutathione S-transferase(GST). Such fusion proteins may be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems ofteninclude enterokinase, thrombin or factor Xa protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety at will, as may be useful in purification or other applications.Other examples are fusion proteins comprising hTRT and the E. coliMaltose Binding Protein (MBP) or E. Coli thioredoxin. Illustrativeexamples of hTRT expression constructs useful in bacterial cells areprovided in Example 6, infra.

[0248] The invention further provides hTRT polypeptides expressed infungal systems, such as Dictyostelium and, preferably, yeast, such asSaccharomyces cerevisiae, Pichia pastoris, Torulopsis holmil,Saccharomyces fragilis, Saccharomyces lactis, Hansenula polymorpha andCandida pseudotropicalis. When hTRT is expressed in yeast, a number ofsuitable vectors are available, including plasmid and yeast artificialchromosomes (YACs) vectors. The vectors typically include expressioncontrol sequences, such as constitutive or inducible promoters (e.g.,such as alpha factor, alcohol oxidase, PGH, and 3-phosphoglyceratekinase or other glycolytic enzymes), and an origin of replication,termination sequences and the like, as desired. Suitable vectors for usein Pichia include pPICZ, His6/pPICZB, pPICZalpha, pPIC3.5K, pPIC9K,pA0815, pGAP2A, B & C, pGAP2alpha A, B, and C (Invitrogen, San Diego,Calif.) and numerous others known in the art or to be developed. In oneembodiment, the vector His6/pPICZB (Invitrogen, San Diego, Calif.) isused to express a His₆-hTRT fusion protein in the yeast Pichia pastoris.An example of a vector useful in Saccharomyces is pYES2 (Invitrogen, SanDiego, Calif.). Illustrative examples of hTRT expression constructsuseful in yeast are provided in Example 6, infra.

[0249] The hTRT polypeptides of the invention may also be expressed inplant cell systems transfected with plant or plant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid). In cases where plant virus expression vectors are used,the expression of an hTRT-encoding sequence may be driven by any of anumber of promoters. For example, viral promoters such as the 35S and19S promoters of CaMV (Brisson et al., 1984, Nature 310:511-514) may beused alone or in combination with the omega leader sequence from TMV(Takamatsu et al., 1987, EMBO J., 6:307-311). Alternatively, plantpromoters such as that from the small subunit gene of RUBISCO (Coruzziet al., 1984, EMBO J., 3:1671-1680; Broglie et al., 1984, Science224:838-843) or heat shock promoters (Winter and Sinibaldi, 1991,Results Probl. Cell Differ., 17:85), or storage protein gene promotersmay be used. These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection (for reviewsof such techniques, see Hobbs or Murry, 1992, in McGRAW HILL YEARBOOK OFSCIENCE AND TECHNOLOGY McGraw Hill New York N.Y., pp. 191-196 [1992]; orWeissbach and Weissbach, 1988, METHODS FOR PLANT MOLECULAR BIOLOGY,Academic Press, New York N.Y., pp. 421-463).

[0250] Another expression system provided by the invention forexpression of hTRT protein is an insect system. A preferred system usesa baculovirus polyhedrin promoter. In one such system, Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. The sequence encoding the gene of interest may be cloned into anonessential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe sequence, e.g., encoding the hTRT protein, will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses are then used to infect S. frugiperdacells or Trichoplusia larvae, in which the hTRT sequence is thenexpressed (see, for general methods, Smith et al., J. Virol., 46:584[1983]; Engelhard et al., Proc. Natl. Acad. Sci. 91:3224-7 [1994]).Useful vectors for baculovirus expression include pBlueBacHis2 A, B & C,pBlueBac4.5, pMelBacB and numerous others known in the art or to bedeveloped. Illustrative examples of hTRT expression constructs useful ininsect cells are provided in Example 6, infra.

[0251] The present invention also provides expression systems in mammalsand mammalian cells. As noted supra, hTRT polynucleotides may beexpressed in mammalian cells (e.g., human cells) for production ofsignificant quantities of hTRT polypeptides (e.g., for purification) orto change the phenotype of a target cell (e.g., for purposes of genetherapy, cell immortalization, or other). In the latter case, the hTRTpolynucleotide expressed may or may not encode a polypeptide with atelomerase catalytic activity. That is, expression may be of a sense orantisense polynucleotide, an inhibitory or stimulatory polypeptide, apolypeptide with zero, one or more telomerase activities, and othercombinations and variants disclosed herein or apparent to one of skillupon review of this disclosure.

[0252] Suitable mammalian host tissue culture cells for expressing thenucleic acids of the invention include any normal mortal or normal orabnormal immortal animal or human cell, including: monkey kidney CV1line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidneyline (293; Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamsterkidney cells (BHK, ATCC CCL 10); CHO (ATCC CCL 61 and CRL 9618); mousesertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HeLa, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (WI38, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TR1 cells (Mather, et al., Annals N.Y. Acad. Sci. 383:44-46(1982); MDCK cells (ATCC CCL 34 and CRL 6253); HEK 293 cells (ATCC CRL1573); and WI-38 cells (ATCC CCL 75; ATCC: American Type CultureCollection, Rockville, Md.). The use of mammalian tissue cell culture toexpress polypeptides is discussed generally in Winnacker, FROM GENES TOCLONES (VCH Publishers, N.Y., N.Y., 1987).

[0253] For mammalian host cells, viral-based and nonviral expressionsystems are provided. Nonviral vectors and systems include plasmids andepisomal vectors, typically with an expression cassette for expressing aprotein or RNA, and human artificial chromosomes (see, e.g., Harringtonet al., 1997, Nat Genet 15:345). For example, nonviral vectors usefulfor expression of hTRT polynucleotides and polypeptides in mammalian(e.g., human) cells include pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen,San Diego Calif.), MPSV vectors, others described in the Invitrogen 1997Catalog (Invitrogen Inc, San Diego Calif.), which is incorporated in itsentirety herein, and numerous others known in the art for otherproteins. Illustrative examples of hTRT expression constructs useful inmammalian cells are provided in Example 6, infra.

[0254] Useful viral vectors include vectors based on retroviruses,adenoviruses, adenoassociated viruses, herpes viruses, vectors based onSV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectorsand Semliki Forest virus (SFV). SFV and vaccinia vectors are discussedgenerally in Ausubel et al., supra, Ch 16. These vectors are often madeup of two components, a modified viral genome and a coat structuresurrounding it (see generally Smith, 1995, Annu. Rev. Microbiol. 49:807), although sometimes viral vectors are introduced in naked form orcoated with proteins other than viral proteins. However, the viralnucleic acid in a vector may be changed in many ways, for example, whendesigned for gene therapy. The goals of these changes are to disablegrowth of the virus in target cells while maintaining its ability togrow in vector form in available packaging or helper cells, to providespace within the viral genome for insertion of exogenous DNA sequences,and to incorporate new sequences that encode and enable appropriateexpression of the gene of interest. Thus, vector nucleic acids generallycomprise two components: essential cis-acting viral sequences forreplication and packaging in a helper line and the transcription unitfor the exogenous gene. Other viral functions are expressed in trans ina specific packaging or helper cell line. Adenoviral vectors (e.g., foruse in human gene therapy) are described in, e.g., Rosenfeld et al.,1992, Cell 68: 143; PCT publications WO 94/12650; 94/12649; and94/12629. In cases where an adenovirus is used as an expression vector,a sequence encoding hTRT may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a nonessential E1 or E3 regionof the viral genome will result in a viable virus capable of expressingin infected host cells (Logan and Shenk, 1984, Proc. Natl. Acad. Sci.,81:3655). Replication-defective retroviral vectors harboring atherapeutic polynucleotide sequence as part of the retroviral genome aredescribed in, e.g., Miller et al., 1990, Mol. Cell. Biol. 10: 4239;Kolberg, 1992, J. NIH Res. 4: 43; and Cornetta et al., 1991, Hum. GeneTher. 2: 215.

[0255] In mammalian cell systems, promoters from mammalian genes or frommammalian viruses are often appropriate. Suitable promoters may beconstitutive, cell type-specific, stage-specific, and/or modulatable orregulatable (e.g., by hormones such as glucocorticoids). Usefulpromoters include, but are not limited to, the metallothionein promoter,the constitutive adenovirus major late promoter, thedexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIIIpromoter, the constitutive MPSV promoter, the tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter), theconstitutive CMV promoter, and promoter-enhancer combinations known inthe art.

[0256] Other regulatory elements may also be required or desired forefficient expression of an hTRT polynucleotide and/or translation of asequence encoding hTRT proteins. For translation, these elementstypically include an ATG initiation codon and adjacent ribosome bindingsite or other sequences. For sequences encoding the hTRT protein,provided its initiation codon and upstream promoter sequences areinserted into an expression vector, no additional translational or othercontrol signals may be needed. However, in cases where only codingsequence, or a portion thereof, is inserted, exogenous transcriptionaland/or translational control signals (e.g., the promoter,ribosome-binding site, and ATG initiation codon) must often be provided.Furthermore, the initiation codon must typically be in the correctreading frame to ensure translation of the desired protein. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. In addition, the efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (Scharf et al., 1994, Results Probl. Cell Differ.20:125; and Bittner et al. 1987, Meth. Enzymol., 153:516). For example,the SV40 enhancer or CMV enhancer may be used to increase expression inmammalian host cells.

[0257] Expression of hTRT gene products can also by effected (increased)by activation of an hTRT promoter or enhancer in a cell such as a humancell, e.g., a telomerase-negative cell line. Activation can be carriedout in a variety of ways, including administration of an exogenouspromoter activating agent, or inhibition of a cellular component thatsuppresses expression of the hTRT gene. It will be appreciated that,conversely, inhibition of promoter function, as described infra, willreduce hTRT gene expression.

[0258] The invention provides inducible and repressible expression ofhTRT polypeptides using such system as the Ecdysone-Inducible ExpressionSystem (Invitrogen), and the Tet-On and Tet-off tetracycline regulatedsystems from Clontech. The ecdysone-inducible expression system uses thesteroid hormone ecdysone analog, muristerone A, to activate expressionof a recombinant protein via a heterodimeric nuclear receptor (No etal., 1996, Proc. Natl. Acad. Sci. USA 93:3346). In one embodiment of theinvention, hTRT is cloned in the pIND vector (Clontech), which containsfive modified ecdysone response elements (E/GREs) upstream of a minimalheat shock promoter and the multiple cloning site. The construct is thentransfected in cell lines stably expressing the ecdysone receptor. Aftertransfection, cells are treated with muristerone A to induceintracellular expression from pIND. In another embodiment of theinvention, hTRT polypeptide is expressed using the Tet-on and Tet-offexpression systems (Clontech) to provide regulated, high-level geneexpression (Gossen et al., 1992, Proc. Natl. Acad. Sci. USA 89:5547;Gossen et al., 1995, Science 268:1766).

[0259] The hTRT vectors of the invention may be introduced into a cell,tissue, organ, patient or animal by a variety of methods. The nucleicacid expression vectors (typically dsDNA) of the invention can betransferred into the chosen host cell by well-known methods such ascalcium chloride transformation (for bacterial systems),electroporation, calcium phosphate treatment, liposome-mediatedtransformation, injection and microinjection, ballistic methods,virosomes, immunoliposomes, polycation:nucleic acid conjugates, nakedDNA, artificial virions, fusion to the herpes virus structural proteinVP22 (Elliot and O'Hare, Cell 88:223), agent-enhanced uptake of DNA, andex vivo transduction. Useful liposome-mediated DNA transfer methods aredescribed in U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S.Pat. No. 4,897,355; PCT publications WO 91/17424, WO 91/16024; Wang andHuang, 1987, Biochem. Biophys. Res. Commun. 147: 980; Wang and Huang,1989, Biochemistry 28: 9508; Litzinger and Huang, 1992, Biochem.Biophys. Acta 1113:201; Gao and Huang, 1991, Biochem. Biophys. Res.Commun. 179: 280. Immunoliposomes have been described as carriers ofexogenous polynucleotides (Wang and Huang, 1987, Proc. Natl. Acad. Sci.U.S.A. 84:7851; Trubetskoy et al., 1992, Biochem. Biophys. Acta1131:311) and may have improved cell type specificity as compared toliposomes by virtue of the inclusion of specific antibodies whichpresumably bind to surface antigens on specific cell types. Behr et al.,1989, Proc. Natl. Acad. Sci. U.S.A. 86:6982 report using lipopolyamineas a reagent to mediate transfection itself, without the necessity ofany additional phospholipid to form liposomes. Suitable delivery methodswill be selected by practitioners in view of acceptable practices andregulatory requirements (e.g., for gene therapy or production of celllines for expression of recombinant proteins). It will be appreciatedthat the delivery methods listed above may be used for transfer ofnucleic acids into cells for purposes of gene therapy, transfer intotissue culture cells, and the like.

[0260] For long-term, high-yield production of recombinant proteins,stable expression will often be desired. For example, cell lines whichstably express hTRT can be prepared using expression vectors of theinvention which contain viral origins of replication or endogenousexpression elements and a selectable marker gene. Following theintroduction of the vector, cells may be allowed to grow for 1-2 days inan enriched media before they are switched to selective media. Thepurpose of the selectable marker is to confer resistance to selection,and its presence allows growth of cells which successfully express theintroduced sequences in selective media. Resistant, stably transfectedcells can be proliferated using tissue culture techniques appropriate tothe cell type. An amplification step, e.g., by administration ofmethyltrexate to cells transfected with a DHFR gene according to methodswell known in the art, can be included.

[0261] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,phosphorylation, lipidation and acylation. Post-translational processingmay also be important for correct insertion, folding and/or function.Different host cells have cellular machinery and characteristicmechanisms specific for each cell for such post-translational activitiesand so a particular cell may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

[0262] As noted supra, when expressing an hTRT protein (includingvariants) in cells or organisms it is sometimes desirable to use an hTRTprotein-encoding polynucleotide that employs a codon distribution otherthan that found in a naturally occurring hTRT gene. hTRTprotein-encoding polynucleotides with alternative codons throughout, orat specific sites, in the coding sequence are used to optimize (e.g.,increase) expression of the hTRT protein in cells, especially non-humancells (e.g., bacterial, plant, fungal, and non-human animal cells) whichhave different preferential codon usage than human cells. Codon changesmay also be used to facilitate manipulation of the hTRT polynucleotide(e.g., by engineering useful tags or restriction sites into the codingsequence), and for other reasons. When the goal is to optimizeexpression (e.g., by increasing translational efficiency), tables ofpreferred codon usage, which are publicly available and are well knownto those of skill, are used to design a suitable polynucleotide by“reverse translation” of the desired (e.g., hTRT) amino acid sequence.Alternatively, preferred codon usage can be determined for a particularorganism (e.g., Pichia pastoris) or class of genes (e.g., highlyexpressed genes of a particular organism) by comparison of publishedgene sequences for the target organism or gene class.

[0263] Illustrative hTRT-encoding polynucleotide sequences are providedin Table 9 (A-E), infra. All of the sequences in Table 9 are in the5′→3′. Table 9A shows an hTRT protein encoding polynucleotide that usesa codon distribution preferentially employed in the bacterium E. coli.Table 9B shows a second polynucleotide sequence particularly useful forexpression in E. coli (and other enteric bacteria) using codonspreferentially used in highly expressed genes in enteric bacteria. Table4C shows an hTRT protein encoding polynucleotide that uses a codondistribution preferentially employed in yeast (i.e., S. cerevisiae).Table 4D shows an hTRT protein encoding polynucleotide that uses a codondistribution preferentially used in highly expressed genes in yeast.Table 4E shows an hTRT protein encoding polynucleotide that uses a“generic” codon distribution that should be efficiently expressed inboth bacteria (e.g., E. coli) and yeast (e.g., S. pombe, S. cerevisiae,P. pastoris) and some insect (e.g., S. frugiperda) cells. Such “generic”polynucleotide sequences (optimized for more than one organism) areuseful for, for example, comparative studies, screening in differentorganisms of hTRT binding or modulatory agents, creation of shuttlevectors, and other uses. In this “generic” sequence, the codon TCT(serine) may not be optimal for expression in Drosophila cells.Therefore, in an alternative embodiment the sequence in Table 4E ismodified to replace TCT with TCC for efficient expression in Drosophilaas well as bacteria and yeast.

Table 9 hTRT-Encoding Polynucleotide Sequences Employing AlternativeCodon Distributions

[0264] TABLE 9A E. coli (all genes) (SEQ ID NO:638) ATG CCG CGC GCG CCGCGC TGC CGC GCG GTG CGC AGC CTG CTG CGC AGC CAT TAT CGC GAA GTG CTG CCGCTG GCG ACC TTT GTG CGC CGC CTG GGC CCG CAG GGC TGG CGC CTG GTG CAG CGCGGC GAT CCG GCG GCG TTT CGC GCG CTG GTG GCG CAG TGC CTG GTG TGC GTG CCGTGG GAT GCG CGC CCG CCG CCG GCG GCG CCG AGC TTT CGC CAG GTG AGC TGC CTGAAA GAA CTG GTG GCG CGC GTG CTG CAG CGC CTG TGC GAA CGC GGC GCG AAA AACGTG CTG GCG TTT GGC TTT GCG CTG CTG GAT GGC GCG CGC GGC GGC CCG CCG GAAGCG TTT ACC ACC AGC GTG CGC AGC TAT CTG CCG AAC ACC GTG ACC GAT GCG CTGCGC GGC AGC GGC GCG TGG GGC CTG CTG CTG CGC CGC GTG GGC GAT GAT GTG CTGGTG CAT CTG CTG GCG CGC TGC GCG CTG TTT GTG CTG GTG GCG CCG AGC TGC GCGTAT CAG GTG TGC GGC CCG CCG CTG TAT CAG CTG GGC GCG GCG ACC CAG GCG CGCCCG CCG CCG CAT GCG AGC GGC CCG CGC CGC CGC CTG GGC TGC GAA CGC GCG TGGAAC CAT AGC GTG CGC GAA GCG GGC GTG CCG CTG GGC CTG CCG GCG CCG GGC GCGCGC CGC CGC GGC GGC AGC GCG AGC CGC AGC CTG CCG CTG CCG AAA CGC CCG CGCCGC GGC GCG GCG CCG GAA CCG GAA CGC ACC CCG GTG GGC CAG GGC AGC TGG GCGCAT CCG GGC CGC ACC CGC GGC CCG AGC GAT CGC GGC TTT TGC GTG GTG AGC CCGGCG CGC CCG GCG GAA GAA GCG ACC AGC CTG GAA GGC GCG CTG AGC GGC ACC CGCCAT AGC CAT CCG AGC GTG GGC CGC CAG CAT CAT GCG GGC CCG CCG AGC ACC AGCCGC CCG CCG CGC CCG TGG GAT ACC CCG TGC CCG CCG GTG TAT GCG GAA ACC AAACAT TTT CTG TAT AGC AGC GGC GAT AAA GAA CAG CTG CGC CCG AGC TTT CTG CTGAGC AGC CTG CGC CCG AGC CTG ACC GGC GCG CGC CGC CTG GTG GAA ACC ATT TTTCTG GGC AGC CGC CCG TGG ATG CCG GGC ACC CCG CGC CGC CTG CCG CGC CTG CCGCAG CGC TAT TGG CAG ATG CGC CCG CTG TTT CTG GAA CTG CTG GGC AAC CAT GCGCAG TGC CCG TAT GGC GTG CTG CTG AAA ACC CAT TGC CCG CTG CGC GCG GCG GTGACC CCG GCG GCG GGC GTG TGC GCG CGC GAA AAA CCG CAG GGC AGC GTG GCG GCGCCG GAA GAA GAA GAT ACC GAT CCG CGC CGC CTG GTG CAG CTG CTG CGC CAG CATAGC AGC CCG TGG CAG GTG TAT GGC TTT GTG CGC GCG TGC CTG CGC CGC CTG GTGCCG CCG GGC CTG TGG GGC AGC CGC CAT AAC GAA CGC CGC TTT CTG CGC AAC ACCAAA AAA TTT ATT AGC CTG GGC AAA CAT GCG AAA CTG AGC CTG CAG GAA CTG ACCTGG AAA ATG AGC GTG CGC GAT TGC GCG TGG CTG CGC CGC AGC CCG GGC GTG GGCTGC GTG CCG GCG GCG GAA CAT CGC CTG CGC GAA GAA ATT CTG GCG AAA TTT CTGCAT TGG CTG ATG AGC GTG TAT GTG GTG GAA CTG CTG CGC AGC TTT TTT TAT GTGACC GAA ACC ACC TTT CAG AAA AAC CGC CTG TTT TTT TAT CGC AAA AGC GTG TGGAGC AAA CTG CAG AGC ATT GGC ATT CGC CAG CAT CTG AAA CGC GTG CAG CTG CGCGAA CTG AGC GAA GCG GAA GTG CGC CAG CAT CGC GAA GCG CGC CCG GCG CTG CTGACC AGC CGC CTG CGC TTT ATT CCG AAA CCG GAT GGC CTG CGC CCG ATT GTG AACATG GAT TAT GTG GTG GGC GCG CGC ACC TTT CGC CGC GAA AAA CGC GCG GAA CGCCTG ACC AGC CGC GTG AAA GCG CTG TTT AGC GTG CTG AAC TAT GAA CGC GCG CGCCGC CCG GGC CTG CTG GGC GCG AGC GTG CTG GGC CTG GAT GAT ATT CAT CGC GCGTGG CGC ACC TTT GTG CTG CGC GTG CGC GCG CAG GAT CCG CCG CCG GAA CTG TATTTT GTG AAA GTG GAT GTG ACC GGC GCG TAT GAT ACC ATT CCG CAG GAT CGC CTGACC GAA GTG ATT GCG AGC ATT ATT AAA CCG CAG AAC ACC TAT TGC GTG CGC CGCTAT GCG GTG GTG CAG AAA GCG GCG CAT GGC CAT GTG CGC AAA GCG TTT AAA AGCCAT GTG AGC ACC CTG ACC GAT CTG CAG CCG TAT ATG CGC CAG TTT GTG GCG CATCTG CAG GAA ACC AGC CCG CTG CGC GAT GCG GTG GTG ATT GAA CAG AGC AGC AGCCTG AAC GAA GCG AGC AGC GGC CTG TTT GAT GTG TTT CTG CGC TTT ATG TGC CATCAT GCG GTG CGC ATT CGC GGC AAA AGC TAT GTG CAG TGC CAG GGC ATT CCG CAGGGC AGC ATT CTG AGC ACC CTG CTG TGC AGC CTG TGC TAT GGC GAT ATG GAA AACAAA CTG TTT GCG GGC ATT CGC CGC GAT GGC CTG CTG CTG CGC CTG GTG GAT GATTTT CTG CTG GTG ACC CCG CAT CTG ACC CAT GCG AAA ACC TTT CTG CGC ACC CTGGTG CGC GGC GTG CCG GAA TAT GGC TGC GTG GTG AAC CTG CGC AAA ACC GTG GTGAAC TTT CCG GTG GAA GAT GAA GCG CTG GGC GGC ACC GCG TTT GTG CAG ATG CCGGCG CAT GGC CTG TTT CCG TGG TGC GGC CTG CTG CTG GAT ACC CGC ACC CTG GAAGTG CAG AGC GAT TAT AGC AGC TAT GCG CGC ACC AGC ATT CGC GCG AGC CTG ACCTTT AAC CGC GGC TTT AAA GCG GGC CGC AAC ATG CGC CGC AAA CTG TTT GGC GTGCTG CGC CTG AAA TGC CAT AGC CTG TTT CTG GAT CTG CAG GTG AAC AGC CTG CAGACC GTG TGC ACC AAC ATT TAT AAA ATT CTG CTG CTG CAG GCG TAT CGC TTT CATGCG TGC GTG CTG CAG CTG CCG TTT CAT CAG CAG GTG TGG AAA AAC CCG ACC TTTTTT CTG CGC GTG ATT AGC GAT ACC GCG AGC CTG TGC TAT AGC ATT CTG AAA GCGAAA AAC GCG GGC ATG AGC CTG GGC GCG AAA GGC GCG GCG GGC CCG CTG CCG AGCGAA GCG GTG CAG TGG CTG TGC CAT CAG GCG TTT CTG CTG AAA CTG ACC CGC CATCGC GTG ACC TAT GTG CCG CTG CTG GGC AGC CTG CGC ACC GCG CAG ACC CAG CTGAGC CGC AAA CTG CCG GGC ACC ACC CTG ACC GCG CTG GAA GCG GCG GCG AAC CCGGCG CTG CCG AGC GAT TTT AAA ACC ATT CTG GAT

[0265] TABLE 9B Enteric Bacteria (High Expressing Genes) (SEQ ID NO:639)1 ATGCCGCGTG CTCCGCGTTG CCGTGCTGTT CGTTCCCTGC TGCGTTCCCA 51 CTACCGTGAAGTTCTGCCGC TGGCTACCTT CGTTCGTCGT CTGGGTCCGC 101 AGGGTTGGCG TCTGGTTCAGCGTGGTGACC CGGCTGCTTT CCGTGCTCTG 151 GTTGCTCAGT GCCTGGTTTG CGTTCCGTGGGACGCTCGTC CGCCGCCGGC 201 TGCTCCGTCC TTCCGTCAGG TTTCCTGCCT GAAAGAACTGGTTGCTCGTG 251 TTCTGCAGCG TCTGTGCGAA CGTGGTGCTA AAAACGTTCT GGCTTTCGGT301 TTCGCTCTGC TGGACGGTGC TCGTGGTGGT CCGCCGGAAG CTTTCACCAC 351CTCCGTTCGT TCCTACCTGC CGAACACCGT TACCGACGCT CTGCGTGGTT 401 CCGGTGCTTGGGGTCTGCTG CTGCGTCGTG TTGGTGACGA CGTTCTGGTT 451 CACCTGCTGG CTCGTTGCGCTCTGTTCGTT CTGGTTGCTC CGTCCTGCGC 501 TTACCAGGTT TGCGGTCCGC CGCTGTACCAGCTGGGTGCT GCTACCCAGG 551 CTCGTCCGCC GCCGCACGCT TCCGGTCCGC GTCGTCGTCTGGGTTGCGAA 601 CGTGCTTGGA ACCACTCCGT TCGTGAAGCT GGTGTTCCGC TGGGTCTGCC651 GGCTCCGGGT GCTCGTCGTC GTGGTGGTTC CGCTTCCCGT TCCCTGCCGC 701TGCCGAAACG TCCGCGTCGT GGTGCTGCTC CGGAACCGGA ACGTACCCCG 751 GTTGGTCAGGGTTCCTGGGC TCACCCGGGT CGTACCCGTG GTCCGTCCGA 801 CCGTGGTTTC TGCGTTGTTTCCCCGGCTCG TCCGGCTGAA GAAGCTACCT 851 CCCTGGAAGG TGCTCTGTCC GGTACCCGTCACTCCCACCC GTCCGTTGGT 901 CGTCAGCACC ACGCTGGTCC GCCGTCCACC TCCCGTCCGCCGCGTCCGTG 951 GGACACCCCG TGCCCGCCGG TTTACGCTGA AACCAAACAC TTCCTGTACT1001 CCTCCGGTGA CAAAGAACAG CTGCGTCCGT CCTTCCTGCT GTCCTCCCTG 1051CGTCCGTCCC TGACCGGTGC TCGTCGTCTG GTTGAAACCA TCTTCCTGGG 1101 TTCCCGTCCGTGGATGCCGG GTACCCCGCG TCGTCTGCCG CGTCTGCCGC 1151 AGCGTTACTG GCAGATGCGTCCGCTGTTCC TGGAACTGCT GGGTAACCAC 1201 GCTCAGTGCC CGTACGGTGT TCTGCTGAAAACCCACTGCC CGCTGCGTGC 1251 TGCTGTTACC CCGGCTGCTG GTGTTTGCGC TCGTGAAAAACCGCAGGGTT 1301 CCGTTGCTGC TCCGGAAGAA GAAGACACCG ACCCGCGTCG TCTGGTTCAG1351 CTGCTGCGTC AGCACTCCTC CCCGTGGCAG GTTTACGGTT TCGTTCGTGC 1401TTGCCTGCGT CGTCTGGTTC CGCCGGGTCT GTGGGGTTCC CGTCACAACG 1451 AACGTCGTTTCCTGCGTAAC ACCAAAAAAT TCATCTCCCT GGGTAAACAC 1501 GCTAAACTGT CCCTGCAGGAACTGACCTGG AAAATGTCCG TTCGTGACTG 1551 CGCTTGGCTG CGTCGTTCCC CGGGTGTTGGTTGCGTTCCG GCTGCTGAAC 1601 ACCGTCTGCG TGAAGAAATC CTGGCTAAAT TCCTGCACTGGCTGATGTCC 1651 GTTTACGTTG TTGAACTGCT GCGTTCCTTC TTCTACGTTA CCGAAACCAC1701 CTTCCAGAAA AACCGTCTGT TCTTCTACCG TAAATCCGTT TGGTCCAAAC 1751TGCAGTCCAT CGGTATCCGT CAGCACCTGA AACGTGTTCA GCTGCGTGAA 1801 CTGTCCGAAGCTGAAGTTCG TCAGCACCGT GAAGCTCGTC CGGCTCTGCT 1851 GACCTCCCGT CTGCGTTTCATCCCGAAACC GGACGGTCTG CGTCCGATCG 1901 TTAACATGGA CTACGTTGTT GGTGCTCGTACCTTCCGTCG TGAAAAACGT 1951 GCTGAACGTC TGACCTCCCG TGTTAAAGCT CTGTTCTCCGTTCTGAACTA 2001 CGAACGTGCT CGTCGTCCGG GTCTGCTGGG TGCTTCCGTT CTGGGTCTGG2051 ACGACATCCA CCGTGCTTGG CGTACCTTCG TTCTGCGTGT TCGTGCTCAG 2101GACCCGCCGC CGGAACTGTA CTTCGTTAAA GTTGACGTTA CCGGTGCTTA 2151 CGACACCATCCCGCAGGACC GTCTGACCGA AGTTATCGCT TCCATCATCA 2201 AACCGCAGAA CACCTACTGCGTTCGTCGTT ACGCTGTTGT TCAGAAAGCT 2251 GCTCACGGTC ACGTTCGTAA AGCTTTCAAATCCCACGTTT CCACCCTGAC 2301 CGACCTGCAG CCGTACATGC GTCAGTTCGT TGCTCACCTGCAGGAAACCT 2351 CCCCGCTGCG TGACGCTGTT GTTATCGAAC AGTCCTCCTC CCTGAACGAA2401 GCTTCCTCCG GTCTGTTCGA CGTTTTCCTG CGTTTCATGT GCCACCACGC 2451TGTTCGTATC CGTGGTAAAT CCTACGTTCA GTGCCAGGGT ATCCCGCAGG 2501 GTTCCATCCTGTCCACCCTG CTGTGCTCCC TGTGCTACGG TGACATGGAA 2551 AACAAACTGT TCGCTGGTATCCGTCGTGAC GGTCTGCTGC TGCGTCTGGT 2601 TGACGACTTC CTGCTGGTTA CCCCGCACCTGACCCACGCT AAAACCTTCC 2651 TGCGTACCCT GGTTCGTGGT GTTCCGGAAT ACGGTTGCGTTGTTAACCTG 2701 CGTAAAACCG TTGTTAACTT CCCGGTTGAA GACGAAGCTC TGGGTGGTAC2751 CGCTTTCGTT CAGATGCCGG CTCACGGTCT GTTCCCGTGG TGCGGTCTGC 2801TGCTGGACAC CCGTACCCTG GAAGTTCAGT CCGACTACTC CTCCTACGCT 2851 CGTACCTCCATCCGTGCTTC CCTGACCTTC AACCGTGGTT TCAAAGCTGG 2901 TCGTAACATG CGTCGTAAACTGTTCGGTGT TCTGCGTCTG AAATGCCACT 2951 CCCTGTTCCT GGACCTGCAG GTTAACTCCCTGCAGACCGT TTGCACCAAC 3001 ATCTACAAAA TCCTGCTGCT GCAGGCTTAC CGTTTCCACGCTTGCGTTCT 3051 GCAGCTGCCG TTCCACCAGC AGGTTTGGAA AAACCCGACC TTCTTCCTGC3101 GTGTTATCTC CGACACCGCT TCCCTGTGCT ACTCCATCCT GAAAGCTAAA 3151AACGCTGGTA TGTCCCTGGG TGCTAAAGGT GCTGCTGGTC CGCTGCCGTC 3201 CGAAGCTGTTCAGTGGCTGT GCCACCAGGC TTTCCTGCTG AAACTGACCC 3251 GTCACCGTGT TACCTACGTTCCGCTGCTGG GTTCCCTGCG TACCGCTCAG 3301 ACCCAGCTGT CCCGTAAACT GCCGGGTACCACCCTGACCG CTCTGGAAGC 3351 TGCTGCTAAC CCGGCTCTGC CGTCCGACTT CAAAACCATCCTGGAC

[0266] TABLE 9C Yeast (All Genes) (SEQ ID NO:640) ATG CCA AGA GCT CCAAGA TGT AGA GCT GTT AGA TCT TTG TTG AGA TCT CAT TAT AGA GAA GTT TTG CCATTG GCT ACT TTT GTT AGA AGA TTG GGT CCA CAA GGT TGG AGA TTG GTT CAA AGAGGT GAT CCA GCT GCT TTT AGA GCT TTG GTT GCT CAA TGT TTG GTT TGT GTT CCATGG GAT GCT AGA CCA CCA CCA GCT GCT CCA TCT TTT AGA CAA GTT TCT TGT TTGAAA GAA TTG GTT GCT AGA GTT TTG CAA AGA TTG TGT GAA AGA GGT GCT AAA AATGTT TTG GCT TTT GGT TTT GCT TTG TTG GAT GGT GCT AGA GGT GGT CCA CCA GAAGCT TTT ACT ACT TCT GTT AGA TCT TAT TTG CCA AAT ACT GTT ACT GAT GCT TTGAGA GGT TCT GGT GCT TGG GGT TTG TTG TTG AGA AGA GTT GGT GAT GAT GTT TTGGTT CAT TTG TTG GCT AGA TGT GCT TTG TTT GTT TTG GTT GCT CCA TCT TGT GCTTAT CAA GTT TGT GGT CCA CCA TTG TAT CAA TTG GGT GCT GCT ACT CAA GCT AGACCA CCA CCA CAT GCT TCT GGT CCA AGA AGA AGA TTG GGT TGT GAA AGA GCT TGGAAT CAT TCT GTT AGA GAA GCT GGT GTT CCA TTG GGT TTG CCA GCT CCA GGT GCTAGA AGA AGA GGT GGT TCT GCT TCT AGA TCT TTG CCA TTG CCA AAA AGA CCA AGAAGA GGT GCT GCT CCA GAA CCA GAA AGA ACT CCA GTT GGT CAA GGT TCT TGG GCTCAT CCA GGT AGA ACT AGA GGT CCA TCT GAT AGA GGT TTT TGT GTT GTT TCT CCAGCT AGA CCA GCT GAA GAA GCT ACT TCT TTG GAA GGT GCT TTG TCT GGT ACT AGACAT TCT CAT CCA TCT GTT GGT AGA CAA CAT CAT GCT GGT CCA CCA TCT ACT TCTAGA CCA CCA AGA CCA TGG GAT ACT CCA TGT CCA CCA GTT TAT GCT GAA ACT AAACAT TTT TTG TAT TCT TCT GGT GAT AAA GAA CAA TTG AGA CCA TCT TTT TTG TTGTCT TCT TTG AGA CCA TCT TTG ACT GGT GCT AGA AGA TTG GTT GAA ACT ATT TTTTTG GGT TCT AGA CCA TGG ATG CCA GGT ACT CCA AGA AGA TTG CCA AGA TTG CCACAA AGA TAT TGG CAA ATG AGA CCA TTG TTT TTG GAA TTG TTG GGT AAT CAT GCTCAA TGT CCA TAT GGT GTT TTG TTG AAA ACT CAT TGT CCA TTG AGA GCT GCT GTTACT CCA GCT GCT GGT GTT TGT GCT AGA GAA CCA CCA CAA GGT TCT GTT GCT GCTCCA GAA GAA GAA GAT ACT GAT CCA AGA AGA TTG GTT CAA TTG TTG AGA CAA CATTCT TCT CCA TGG CAA GTT TAT GGT TTT GTT AGA GCT TGT TTG AGA AGA TTG GTTCCA CCA GGT TTG TGG GGT TCT AGA CAT AAT GAA AGA AGA TTT TTG AGA AAT ACTAAA AAA TTT ATT TCT TTG GGT AAA CAT GCT AAA TTG TCT TTG CAA GAA TTG ACTTGG AAA ATG TCT GTT AGA GAT TGT GCT TGG TTG AGA AGA TCT CCA GGT GTT GGTTGT GTT CCA GCT GCT GAA CAT AGA TTG AGA GAA GAA ATT TTG GCT AAA TTT TTGCAT TGG TTG ATG TCT GTT TAT GTT GTT GAA TTG TTG AGA TCT TTT TTT TAT GTTACT GAA ACT ACT TTT CAA AAA AAT AGA TTG TTT TTT TAT AGA AAA TCT GTT TGGTCT AAA TTG CAA TCT ATT GGT ATT AGA CAA CAT TTG AAA AGA GTT CAA TTG AGAGAA TTG TCT GAA GCT GAA GTT AGA CAA CAT AGA GAA GCT AGA CCA GCT TTG TTGACT TCT AGA TTG AGA TTT ATT CCA AAA CCA GAT GGT TTG AGA CCA ATT GTT AATATG GAT TAT GTT GTT GGT GCT AGA ACT TTT AGA AGA GAA AAA AGA GCT GAA AGATTG ACT TCT AGA GTT AAA GCT TTG TTT TCT GTT TTG AAT TAT GAA AGA GCT AGAAGA CCA GGT TTG TTG GGT GCT TCT GTT TTG GGT TTG GAT GAT ATT CAT AGA GCTTGG AGA ACT TTT GTT TTG AGA GTT AGA GCT CAA GAT CCA CCA CCA GAA TTG TATTTT GTT AAA GTT GAT GTT ACT GGT GCT TAT GAT ACT ATT CCA CAA GAT AGA TTGACT GAA GTT ATT GCT TCT ATT ATT AAA CCA CAA AAT ACT TAT TGT GTT AGA AGATAT GCT GTT GTT CAA AAA GCT GCT CAT GGT CAT GTT AGA AAA GCT TTT AAA TCTCAT GTT TCT ACT TTG ACT GAT TTG CAA CCA TAT ATG AGA CAA TTT GTT GCT CATTTG CAA GAA ACT TCT CCA TTG AGA GAT GCT GTT GTT ATT GAA CAA TCT TCT TCTTTG AAT GAA GCT TCT TCT GGT TTG TTT GAT GTT TTT TTG AGA TTT ATG TGT CATCAT GCT GTT AGA ATT AGA GGT AAA TCT TAT GTT CAA TGT CAA GGT ATT CCA CAAGGT TCT ATT TTG TCT ACT TTG TTG TGT TCT TTG TGT TAT GGT GAT ATG GAA AATAAA TTG TTT GCT GGT ATT AGA AGA GAT GGT TTG TTG TTG AGA TTG GTT GAT GATTTT TTG TTG GTT ACT CCA CAT TTG ACT CAT GCT AAA ACT TTT TTG AGA ACT TTGGTT AGA GGT GTT CCA GAA TAT GGT TGT GTT GTT AAT TTG AGA AAA ACT GTT GTTAAT TTT CCA GTT GAA GAT GAA GCT TTG GGT GGT ACT GCT TTT GTT CAA ATG CCAGCT CAT GGT TTG TTT CCA TGG TGT GGT TTG TTG TTG GAT ACT AGA ACT TTG GAAGTT CAA TCT GAT TAT TCT TCT TAT GCT AGA ACT TCT ATT AGA GCT TCT TTG ACTTTT AAT AGA GGT TTT AAA GCT GGT AGA AAT ATG AGA AGA AAA TTG TTT GGT GTTTTG AGA TTG AAA TGT CAT TCT TTG TTT TTG GAT TTG CAA GTT AAT TCT TTG CAAACT GTT TGT ACT AAT ATT TAT AAA ATT TTG TTG TTG CAA GCT TAT AGA TTT CATGCT TGT GTT TTG CAA TTG CCA TTT CAT CAA CAA GTT TGG AAA AAT CCA ACT TTTTTT TTG AGA GTT ATT TCT GAT ACT GCT TCT TTG TGT TAT TCT ATT TTG AAA GCTAAA AAT GCT GGT ATG TCT TTG GGT GCT AAA GGT GCT GCT GGT CCA TTG CCA TCTGAA GCT GTT CAA TGG TTG TGT CAT CAA GCT TTT TTG TTG AAA TTG ACT AGA CATAGA GTT ACT TAT GTT CCA TTG TTG GGT TCT TTG AGA ACT GCT CAA ACT CAA TTGTCT AGA AAA TTG CCA GGT ACT ACT TTG ACT GCT TTG GAA GCT GCT GCT AAT CCAGCT TTG CCA TCT GAT TTT AAA ACT ATT TTG GAT

[0267] TABLE 9D Yeast (High Expressing Genes) (SEQ ID NO:641) ATG CCAAGA GCT CCA AGA TGT AGA GCT GTT AGA TCT TTG TTG AGA TCT CAC TAC AGA GAAGTT TTG CCA TTG GCT ACT TTC GTT AGA AGA TTG GGT CCA CAA GGT TGG AGA TTGGTT CAA AGA GGT GAC CCA GCT GCT TTC AGA GCT TTG GTT GCT CAA TGT TTG GTTTGT GTT CCA TGG GAC GCT AGA CCA CCA CCA GCT GCT CCA TCT TTC AGA CAA GTTTCT TGT TTG AAG GAA TTG GTT GCT AGA GTT TTG CAA AGA TTG TGT GAA AGA GGTGCT AAG AAC GTT TTG GCT TTC GGT TTC GCT TTG TTG GAC GGT GCT AGA GGT GGTCCA CCA GAA GCT TTC ACT ACT TCT GTT AGA TCT TAC TTG CCA AAC ACT GTT ACTGAC GCT TTG AGA GGT TCT GGT GCT TGG GGT TTG TTG TTG AGA AGA GTT GGT GACGAC GTT TTG GTT CAC TTG TTG GCT AGA TGT GCT TTG TTC GTT TTG GTT GCT CCATCT TGT GCT TAC CAA GTT TGT GGT CCA CCA TTG TAC CAA TTG GGT GCT GCT ACTCAA GCT AGA CCA CCA CCA CAC GCT TCT GGT CCA AGA AGA AGA TTG GGT TGT GAAAGA GCT TGG AAC CAC TCT GTT AGA GAA GCT GGT GTT CCA TTG GGT TTG CCA GCTCCA GGT GCT AGA AGA AGA GGT GGT TCT GCT TCT AGA TCT TTG CCA TTG CCA AAGAGA CCA AGA AGA GGT GCT GCT CCA GAA CCA GAA AGA ACT CCA GTT GGT CAA GGTTCT TGG GCT CAC CCA GGT AGA ACT AGA GGT CCA TCT GAC AGA GGT TTC TGT GTTGTT TCT CCA GCT AGA CCA GCT GAA GAA GCT ACT TCT TTG GAA GGT GCT TTG TCTGGT ACT AGA CAC TCT CAC CCA TCT GTT GGT AGA CAA CAC CAC GCT GGT CCA CCATCT ACT TCT AGA CCA CCA AGA CCA TGG GAC ACT CCA TGT CCA CCA GTT TAC GCTGAA ACT AAG CAC TTC TTG TAC TCT TCT GGT GAC AAG GAA CAA TTG AGA CCA TCTTTC TTG TTG TCT TCT TTG AGA CCA TCT TTG ACT GGT GCT AGA AGA TTG GTT GAAACT ATT TTC TTG GGT TCT AGA CCA TGG ATG CCA GGT ACT CCA AGA AGA TTG CCAAGA TTG CCA CAA AGA TAC TGG CAA ATG AGA CCA TTG TTC TTG GAA TTG TTG GGTAAC CAC GCT CAA TGT CCA TAC GGT GTT TTG TTG AAG ACT CAC TGT CCA TTG AGAGCT GCT GTT ACT CCA GCT GCT GGT GTT TGT GCT AGA GAA AAG CCA CAA GGT TCTGTT GCT GCT CCA GAA GAA GAA GAC ACT GAC CCA AGA AGA TTG GTT CAA TTG TTGAGA CAA CAC TCT TCT CCA TGG CAA GTT TAC GGT TTC GTT AGA GCT TGT TTG AGAAGA TTG GTT CCA CCA GGT TTG TGG GGT TCT AGA CAC AAC GAA AGA AGA TTC TTGAGA AAC ACT AAG AAG TTC ATT TCT TTG GGT AAG CAC GCT AAG TTG TCT TTG CAAGAA TTG ACT TGG AAG ATG TCT GTT AGA GAC TGT GCT TGG TTG AGA AGA TCT CCAGGT GTT GGT TGT GTT CCA GCT GCT GAA CAC AGA TTG AGA GAA GAA ATT TTG GCTAAG TTC TTG CAC TGG TTG ATG TCT GTT TAC GTT GTT GAA TTG TTG AGA TCT TTCTTC TAC GTT ACT GAA ACT ACT TTC CAA AAG AAC AGA TTG TTC TTC TAC AGA AAGTCT GTT TGG TCT AAG TTG CAA TCT ATT GGT ATT AGA CAA CAC TTG AAG AGA GTTCAA TTG AGA GAA TTG TCT GAA GCT GAA GTT AGA CAA CAC AGA GAA GCT AGA CCAGCT TTG TTG ACT TCT AGA TTG AGA TTC ATT CCA AAG CCA GAC GGT TTG AGA CCAATT GTT AAC ATG GAC TAC GTT GTT GGT GCT AGA ACT TTC AGA AGA GAA AAG AGAGCT GAA AGA TTG ACT TCT AGA GTT AAG GCT TTG TTC TCT GTT TTG AAC TAC GAAAGA GCT AGA AGA CCA GGT TTG TTG GGT GCT TCT GTT TTG GGT TTG GAC GAC ATTCAC AGA GCT TGG AGA ACT TTC GTT TTG AGA GTT AGA GCT CAA GAC CCA CCA CCAGAA TTG TAC TTC GTT AAG GTT GAC GTT ACT GGT GCT TAC GAC ACT ATT CCA CAAGAC AGA TTG ACT GAA GTT ATT GCT TCT ATT ATT AAG CCA CAA AAC ACT TAC TGTGTT AGA AGA TAC GCT GTT GTT CAA AAG GCT GCT CAC GGT CAC GTT AGA AAG GCTTTC AAG TCT CAC GTT TCT ACT TTG ACT GAC TTG CAA CCA TAC ATG AGA CAA TTCGTT GCT CAC TTG CAA GAA ACT TCT CCA TTG AGA GAC GCT GTT GTT ATT GAA CAATCT TCT TCT TTG AAC GAA GCT TCT TCT GGT TTG TTC GAC GTT TTC TTG AGA TTCATG TGT CAC CAC GCT GTT AGA ATT AGA GGT AAG TCT TAC GTT CAA TGT CAA GGTATT CCA CAA GGT TCT ATT TTG TCT ACT TTG TTG TGT TCT TTG TGT TAC GGT GACATG GAA AAC AAG TTG TTC GCT GGT ATT AGA AGA GAC GGT TTG TTG TTG AGA TTGGTT GAC GAC TTC TTG TTG GTT ACT CCA CAC TTG ACT CAC GCT AAG ACT TTC TTGAGA ACT TTG GTT AGA GGT GTT CCA GAA TAC GGT TGT GTT GTT AAC TTG AGA AAGACT GTT GTT AAC TTC CCA GTT GAA GAC GAA GCT TTG GGT GGT ACT GCT TTC GTTCAA ATG CCA GCT CAC GGT TTG TTC CCA TGG TGT GGT TTG TTG TTG GAC ACT AGAACT TTG GAA GTT CAA TCT GAC TAC TCT TCT TAC GCT AGA ACT TCT ATT AGA GCTTCT TTG ACT TTC AAC AGA GGT TTC AAG GCT GGT AGA AAC ATG AGA AGA AAG TTGTTC GGT GTT TTG AGA TTG AAG TGT CAC TCT TTG TTC TTG GAC TTG CAA GTT AACTCT TTG CAA ACT GTT TGT ACT AAC ATT TAC AAG ATT TTG TTG TTG CAA GCT TACAGA TTC CAC GCT TGT GTT TTG CAA TTG CCA TTC CAC CAA CAA GTT TGG AAG AACCCA ACT TTC TTC TTG AGA GTT ATT TCT GAC ACT GCT TCT TTG TGT TAC TCT ATTTTG AAG GCT AAG AAC GCT GGT ATG TCT TTG GGT GCT AAG GGT GCT GCT GGT CCATTG CCA TCT GAA GCT GTT CAA TGG TTG TGT CAC CAA GCT TTC TTG TTG AAG TTGACT AGA CAC AGA GTT ACT TAC GTT CCA TTG TTG GGT TCT TTG AGA ACT GCT CAAACT CAA TTG TCT AGA AAG TTG CCA GGT ACT ACT TTG ACT GCT TTG GAA GCT GCTGCT AAC CCA GCT TTG CCA TCT GAC TTC AAG ACT ATT TTG GAC

[0268] TABLE 9E “Generic” hTRT Protein Encoding Sequence (SEQ ID NO:642)ATG CCA CGT GCC CCA CGT TGT CGT GCC GTT CGT TCT TTG TTG CGT TCT CAC TACCGT GAA GTT TTG CCA TTG GCC ACC TTC GTT CGT CGT TTG GGT CCA CAA GGT TGGCGT TTG GTT CAA CGT GGT GAT CCA GCC GCC TTC CGT GCC TTG GTT GCC CAA TGTTTG GTT TGT GTT CCA TGG GAT GCC CGT CCA CCA CCA GCC GCC CCA TCT TTC CGTCAA GTT TCT TGT TTG AAA GAA TTG GTT GCC CGT GTT TTG CAA CGT TTG TGT GAACGT GGT GCC AAA AAC GTT TTG GCC TTC GGT TTC GCC TTG TTG GAT GGT GCC CGTGGT GGT CCA CCA GAA GCC TTC ACC ACC TCT GTT CGT TCT TAC TTG CCA AAC ACCGTT ACC GAT GCC TTG CGT GGT TCT GGT GCC TGG GGT TTG TTG TTG CGT CGT GTTGGT GAT GAT GTT TTG GTT CAC TTG TTG GCC CGT TGT GCC TTG TTC GTT TTG GTTGCC CCA TCT TGT GCC TAC CAA GTT TGT GGT CCA CCA TTG TAC CAA TTG GGT GCCGCC ACC CAA GCC CGT CCA CCA CCA CAC GCC TCT GGT CCA CGT CGT CGT TTG GGTTGT GAA CGT GCC TGG AAC CAC TCT GTT CGT GAA GCC GGT GTT CCA TTG GGT TTGCCA GCC CCA GGT GCC CGT CGT CGT GGT GGT TCT GCC TCT CGT TCT TTG CCA TTGCCA AAA CGT CCA CGT CGT GGT GCC GCC CCA GAA CCA GAA CGT ACC CCA GTT GGTCAA GGT TCT TGG GCC CAC CCA GGT CGT ACC CGT GGT CCA TCT GAT CGT GGT TTCTGT GTT GTT TCT CCA GCC CGT CCA GCC GAA GAA GCC ACC TCT TTG GAA GGT GCCTTG TCT GGT ACC CGT CAC TCT CAC CCA TCT GTT GGT CGT CAA CAC CAC GCC GGTCCA CCA TCT ACC TCT CGT CCA CCA CGT CCA TGG GAT ACC CCA TGT CCA CCA GTTTAC GCC GAA ACC AAA CAC TTC TTG TAC TCT TCT GGT GAT AAA GAA CAA TTG CGTCCA TCT TTC TTG TTG TCT TCT TTG CGT CCA TCT TTG ACC GGT GCC CGT CGT TTGGTT QAA ACC ATT TTC TTG GGT TCT CGT CCA TGG ATG CCA GGT ACC CCA CGT CGTTTG CCA CGT TTG CCA CAA CGT TAC TGG CAA ATG CGT CCA TTG TTC TTG GAA TTGTTG GGT AAC CAC GCC CAA TGT CCA TAC GGT GTT TTG TTG AAA ACC CAC TGT CCATTG CGT GCC GCC GTT ACC CCA GCC GCC GGT GTT TGT GCC CGT GAA AAA CCA CAAGGT TCT GTT GCC GCC CCA GAA GAA GAA GAT ACC GAT CCA CGT CGT TTG GTT CAATTG TTG CGT CAA CAC TCT TCT CCA TGG CAA GTT TAC GGT TTC GTT CGT GCC TGTTTG CGT CGT TTG GTT CCA CCA GGT TTG TGG GGT TCT CGT CAC AAC GAA CGT CGTTTC TTG CGT AAC ACC AAA AAA TTC ATT TCT TTG GGT AAA CAC GCC AAA TTG TCTTTG CAA GAA TTG ACC TGG AAA ATG TCT GTT CGT GAT TGT GCC TGG TTG CGT CGTTCT CCA GGT GTT GGT TGT GTT CCA GCC GCC GAA CAC CGT TTG CGT GAA GAA ATTTTG GCC AAA TTC TTG CAC TGG TTG ATG TCT GTT TAC GTT GTT GAA TTG TTG CGTTCT TTC TTC TAC GTT ACC GAA ACC ACC TTC CAA AAA AAC CGT TTG TTC TTC TACCGT AAA TCT GTT TGG TCT AAA TTG CAA TCT ATT GGT ATT CGT CAA CAC TTG AAACGT GTT CAA TTG CGT GAA TTG TCT GAA GCC GAA GTT CGT CAA CAC CGT GAA GCCCGT CCA GCC TTG TTG ACC TCT CGT TTG CGT TTC ATT CCA AAA CCA GAT GGT TTGCGT CCA ATT GTT AAC ATG GAT TAC GTT GTT GGT GCC CGT ACC TTC CGT CGT GAAAAA CGT GCC GAA CGT TTG ACC TCT CGT GTT AAA GCC TTG TTC TCT GTT TTG AACTAC GAA CGT GCC CGT CGT CCA GGT TTG TTG GGT GCC TCT GTT TTG GGT TTG GATGAT ATT CAC CGT GCC TGG CGT ACC TTC GTT TTG CGT GTT CGT GCC CAA GAT CCACCA CCA GAA TTG TAC TTC GTT AAA GTT GAT GTT ACC GGT GCC TAC GAT ACC ATTCCA CAA GAT CGT TTG ACC GAA GTT ATT GCC TCT ATT ATT AAA CCA CAA AAC ACCTAC TGT GTT CGT CGT TAC GCC GTT GTT CAA AAA GCC GCC CAC GGT CAC GTT CGTAAA GCC TTC AAA TCT CAC GTT TCT ACC TTG ACC GAT TTG CAA CCA TAC ATG CGTCAA TTC GTT GCC CAC TTG CAA GAA ACC TCT CCA TTG CGT GAT GCC GTT GTT ATTGAA CAA TCT TCT TCT TTG AAC GAA GCC TCT TCT GGT TTG TTC GAT GTT TTC TTGCGT TTC ATG TGT CAC CAC GCC GTT CGT ATT CGT GGT AAA TCT TAC GTT CAA TGTCAA GGT ATT CCA CAA GGT TCT ATT TTG TCT ACC TTG TTG TGT TCT TTG TGT TACGGT GAT ATG GAA AAC AAA TTG TTC GCC GGT ATT CGT CGT GAT GGT TTG TTG TTGCGT TTG GTT GAT GAT TTC TTG TTG GTT ACC CCA CAC TTG ACC CAC GCC AAA ACCTTC TTG CGT ACC TTG GTT CGT GGT GTT CCA GAA TAC GGT TGT GTT GTT AAC TTGCGT AAA ACC GTT GTT AAC TTC CCA GTT GAA GAT GAA GCC TTG GGT GGT ACC GCCTTC GTT CAA ATG CCA GCC CAC GGT TTG TTC CCA TGG TGT GGT TTG TTG TTG GATACC CGT ACC TTG GAA GTT CAA TCT GAT TAC TCT TCT TAC GCC CGT ACC TCT ATTCGT GCC TCT TTG ACC TTC AAC CGT GGT TTC AAA GCC GGT CGT AAC ATG CGT CGTAAA TTG TTC GGT GTT TTG CGT TTG AAA TGT CAC TCT TTG TTC TTG GAT TTG CAAGTT AAC TCT TTG CAA ACC GTT TGT ACC AAC ATT TAC AAA ATT TTG TTG TTG CAAGCC TAC CGT TTC CAC GCC TGT GTT TTG CAA TTG CCA TTC CAC CAA CAA GTT TGGAAA AAC CCA ACC TTC TTC TTG CGT GTT ATT TCT GAT ACC GCC TCT TTG TGT TACTCT ATT TTG AAA GCC AAA AAC GCC GGT ATG TCT TTG GGT GCC AAA GGT GCC GCCGGT CCA TTG CCA TCT GAA GCC GTT CAA TGG TTG TGT CAC CAA GCC TTC TTG TTGAAA TTG ACC CGT CAC CGT GTT ACC TAC GTT CCA TTG TTG GGT TCT TTG CGT ACCGCC CAA ACC CAA TTG TCT CGT AAA TTG CCA GGT ACC ACC TTG ACC GCC TTG GAAGCC GCC GCC AAC CCA GCC TTG CCA TCT GAT TTC AAA ACC ATT TTG GAT

[0269] Following determination of the desired nucleotide sequence forthe hTRT protein-encoding polynucleotide, the polynucleotide can be madeby any suitable method including de novo chemical synthesis, directedmutagenesis of a synthetic or naturally occurring TRT gene or cDNA, or acombination of these methods. In one exemplary embodiment,oligonucleotides (typically 50-100 bases in length) are synthesized witha 5′ phosphate group and include approximately 10-base overhangs(relative to adjacent oligonucleotides in the assembled gene) to directsubsequent ligations. Following purification and desalting, eacholigonucleotide is annealed to its complement (e.g., by combining pairsof oligonucleotides in equimolar amounts in a neutral pH buffer with50-200 mM NaCl and 0.5 mM MgCl₂). Annealing may be monitored by nativePAGE. The resulting double-stranded oligonucleotides are ligated totheir neighbors in pairs. After each ligation the products aregel-purified, then ligated to the appropriate (neighboring)double-stranded DNAs. In this manner, fragments of approximately 600-800basepairs are built up. These intermediate fragments are then clonedinto vectors and sequenced. The fragments are then combined into asingle vector (resulting in a vector containing a polynucleotide withthe desired hTRT protein-encoding sequence). This step is facilitated byusing restriction sites present in, or engineered into, thepolynucleotide sequence. Alternatively, the fragments can be built up byligation until the complete cDNA is assembled and the assembled sequencecloned into a vector. Numerous other alternative methods and approacheswill be apparent to those of skill in the art.

[0270] Table 10A shows an exemplary set of oligonucleotides that can beused to produce a polynucleotide, shown in Table 10B, that employs acodon distribution preferentially used by highly expressed genes in E.coli. The sequence in Table 5B contains silent changes to some codons tointroduce useful restriction sites spaced every 300-800 base pairs, tofacilitate subcloning and modification. Oligonucleotide pairs for theinitial annealing steps are indicated by the labels “T” (top strand) and“B” (bottom strand). The full-length polynucleotide (Table 10B) encodesthe hTRT protein (with the start codon at nucleotides 28-30) andcontains Sac I and Xho I sites at the termini flanking the open readingframe, which are useful for cloning into a variety of vectors (e.g.,pBluescript II KS, Stratagene Inc., San Diego Calif.). Once cloned intoan appropriate vector, the hTRT sequence may be expressed, modified(e.g., by site directed or cassette mutagenesis), subcloned, orotherwise used or manipulated. In one embodiment, the polynucleotide issubcloned into a pET vector containing a T7 RNA polymerase promoter(Novagen Inc., Madison, Wis.) and introduced into an E. coli strainhaving an inducible T7 polymerase (Novagen Inc., Madison, Wis.). Oneadvantage to the pET system is that the E. coli culture may be grownbefore the T7 RNA polymerase gene is induced, resulting in very highlevels of transcription and minimizing the effect of any potentialdetrimental effect of the expressed protein on the cells.

Table 10 Synthesis of hTRT Polynucleotide Having Alternative CodonDistribution

[0271] TABLE 10A Oligonucleotides (SEQ ID NOS:643-721)  1BCCAGCGGCAGAACTTCGCGATAGTGGGAACGCAGCAGGGAACGAACAGGACGGGAACGCGGAGCACGCGGCATATGGTCGACTCTAGAGCTC CCGCGTGC  1TGCACGCGGGAGCTCTAGAGTCGACCATATGCCGCGTGCTCCGCGTTGCCGTGCTGTTCGTTCCCTGCTGCGTTCCCACTATCGCGAAGTT  2BGGCACTGAGCAACCAGAGCACGGAAAGCAGCCGGGTCACCACGCTGAACCAGACGCCAACCCTGCGGGCCCAGACGACGAACGAAGGTAG  2TCTGCCGCTGGCTACCTTCGTTCGTCGTCTGGGCCCGCAGGGTTGGCGTCTGGTTCAGCGTGGTGACCCGGCTGCTTTCCGTGCTCTGGTT  3BGAACACGAGCAACCAGTTCTTTCAGGCAGGAAACCTGACGGAAGGACGGAGCAGCCGGCGGCGGACGAGCGTCCCACGGAACGCAAACCA  3TGCTCAGTGCCTGGTTTGCGTTCCGTGGGACGCTCGTCCGCCGCCGGCTGCTCCGTCCTTCCGTCAGGTTTCCTGCCTGAAAGAACTGGTT  4BATGCTTCCGGCGGACCACCACGAGCACCGTCCAGCAGAGCGAAACCGAAAGCCAGAACGTTTTTAGCACCACGTTCGCACAGACGCTGCA  4TGCTCGTGTTCTGCAGCGTCTGTGCGAACGTGGTGCTAAAAACGTTCTGGCTTTCGGTTTCGCTCTGCTGGACGGTGCTCGTGGTGGTCCG  5BCAACACGACGCAGCAGCAGACCCCAAGCACCGGAACCACGCAGAGCGTCGGTAACGGTGTTCGGCAGGTAGGAACGAACGGAGGTGGTGA  5TCCGGAAGCATTCACCACCTCCGTTCGTTCCTACCTGCCGAACACCGTTACCGACGCTCTGCGTGGTTCCGGTGCTTGGGGTCTGCTGCTG  6BGCGGCGGACCACAAACCTGGTAAGCGCAGGACGGAGCAACCAGAACGAACAGAGCGCAACGAGCCAGCAGGTGAACGAGAACGTCGTCAC  6TCGTCGTGTTGGTGACGACGTTCTGGTTCACCTGCTGGCTCGTTGCGCTCTGTTCGTTCTGGTTGCTCCGTCCTGCGCTTACCAGGTTTGT  7BGGTTCCAAGCACGTTCGCAACCCAGACGACGACGCGGACCGGAAGCGTGCGGCGGCGGACGAGCCTGGGTAGCAGCACCCAGCTGGTACA  7TGGTCCGCCGCTGTACCAGCTGGGTGCTGCTACCCAGGCTCGTCCGCCGCCGCACGCTTCCGGTCCGCGTCGTCGTCTGGGTTGCGAACGT  8BGCAGCGGCAGGGAACGGGAAGCGGAACCACCACGACGACGAGCACCCGGAGCCGGCAGACCCAGCGGAACACCAGCTTCACGAACGGAGT  8TGCTTGGAACCACTCCGTTCGTGAAGCTGGTGTTCCGCTGGGTCTGCCGGCTCCGGGTGCTCGTCGTCGTGGTGGTTCCGCTTCCCGTTCC  9BGACCACGGGTACGACCCGGGTGAGCCCAGGAACCCTGACCAACCGGGGTACGTTCCGGTTCCGGAGCAGCACCACGACGCGGACGTTTCG  9TCTGCCGCTGCCGAAACGTCCGCGTCGTGGTGCTGCTCCGGAACCGGAACGTACCCCGGTTGGTCAGGGTTCCTGGGCTCACCCGGGTCGT 10BAGTGACGGGTGCCGGACAGAGCACCTTCCAGGGAGGTAGCTTCTTCAGCCGGACGAGCCGGGGAAACAACGCAGAAACCACGGTCGGACG 10TACCCGTGGTCCGTCCGACCGTGGTTTCTGCGTTGTTTCCCCGGCTCGTCCGGCTGAAGAAGCTACCTCCCTGGAAGGTGCTCTGTCCGGC 11BAAACCGGCGGGCACGGGGTGTCCCACGGACGCGGCGGACGGGAGGTGGACGGCGGACCAGCGTGGTGCTGACGACCAACGGACGGGTGGG 11TACCCGTCACTCCCACCCGTCCGTTGGTCGTCAGCACCACGCTGGTCCGCCGTCCACCTCCCGTCCGCCGCGTCCGTGGGACACCCCGTGC 12BTCAGGGACGGACGCAGGGAGGACAGCAGGAAGGACGGACGCAGCTGTTCTTTGTCACCGGAGGAGTACAGGAAGTGTTTGGTTTCAGCGT 12TCCGCCGGTTTACGCTGAAACCAAACACTTCCTGTACTCCTCCGGTGACAAAGAACAGCTGCGTCCGTGCTTCGTGCTGTCCTCCCTGCGT 13BGCTGCGGCAGACGCGGCAGACGACGCGGGGTGCCCGGCATCCACGGACGGGAACCCAGGAAGATAGTTTCAACCAGACGACGAGCACCGG 13TCCGTCCCTGACCGGTGCTCGTCGTCTGGTTGAAACTATCTTCCTGGGTTCCCGTCCGTGGATGCCGGGCACCCCGCGTCGTCTGCCGCGT 14BGCGGGCAGTGGGTTTTCAGCAGAACACCATACGGGCACTGAGCGTGGTTGCCCAGCAGTTCCAGGAACAGCGGACGCATCTGCCAGTAAC 14TCTGCCGCAGCGTTACTGGCAGATGCGTCCGCTGTTCCTGGAACTGCTGGGCAACCACGCTCAGTGCCCGTATGGTGTTCTGCTGAAAACC 15BGGTCGGTATCTTCTTCTTCCGGAGCAGCAACGGAACCCTGCGGTTTTTCACGAGCGCAAACACCAGCAGCCGGGGTAACAGCAGCACGCA 15TCACTGCCCGCTGCGTGCTGCTGTTACCCCGGCTGCTGGTGTTTGCGCTCGTGAAAAACCGCAGGGTTCCGTTGCTGCTCCGGAAGAAGAA 16BGCGGAACCAGACGACGCAGGCATGCACGAACGAAACCGTAAACCTGCCACGGGGAGGAGTGCTGACGCAGCAGCTGAACCAGACGACGCG 16TGATACCGACCCGCGTCGTCTGGTTCAGCTGCTGCGTCAGCACTCCTCCCCGTGGCAGGTTTACGGTTTCGTTCGTGCATGCCTGCGTCGT 17BGGGACAGTTTAGCGTGTTTACCCAGGGAGATGAATTTTTTGGTGTTACGCAGGAAACGACGTTCGTTGTGACGGGAACCCCACAGACCCG 17TCTGGTTCCGCCGGGTCTGTGGGGTTCCCGTCACAACGAACGTCGTTTCCTGCGTAACACCAAAAAATTCATCTCCCTGGGTAAACACGCT 18BGGTGTTCAGCAGCCGGAACGCAACCAACACCCGGAGAACGACGCAGCCAAGCGCAGTCACAACGGACATTTTCCAGGTCAGTTCCTGCA 18TAAACTGTCCCTGCAGGAACTGACCTGGAAAATGTCCGTTCGTGACTGCGCTTGGCTGCGTCGTTCTCCGGGTGTTGGTTGCGTTCCGGCT 19BCGGTAACGTAGAAGAAGGAACGCAGCAGTTCAACAACGTATACGGACATCAGCCAGTGCAGGAATTTAGCCAGGATTTCTTCACGCAGAC 19TGCTGAACACCGTCTGCGTGAAGAAATCCTGGCTAAATTCCTGCACTGGCTGATGTCCGTATACGTTGTTGAACTGCTGCGTTCCTTCTTC 20BGTTTCAGGTGCTGACGGATACCGATGGACTGCAGTTTGGACCAAACGGATTTACGGTAGAAGAACAGACGGTTTTTCTGGAAGGTGGTTT 20TTACGTTACCGAAACCACCTTCCAGAAAAACCGTCTGTTCTTCTACCGTAAATCCGTTTGGTCCAAACTGCAGTCCATCGGTATCCGTCAG 21BGATGAAACGCAGACGGGAGGTCAGCAGAGCCGGACGAGCTTCACGGTGCTGACGAACTTCAGCTTCGGACAGTTCACGCAGCTGAACAC 21TCACCTGAAACGTGTTCAGCTGCGTGAACTGTCCGAAGCTGAAGTTCGTCAGCACCGTGAACTCGTCCGGCTCTGCTGACCTCCCGTCTG 22BTCAGACGCTCAGCACGTTTTTCACGACGGAAGGTACGAGCACCAACAACGTAGTCCATGTTTACGATCGGACGCAGACCGTCCGGTTTCG 22TCGTTTCATCCCGAAACCGGACGGTCTGCGTCCGATCGTAAACATGGACTACGTTGTTGGTGCTCGTACCTTCCGTCGTGAAAAACGTGCT 23BCGTCCAGACCCAGAACGGAAGCACCCAGCAGACCCGGACGACGAGCACGTTCGTAGTTCAGAACGGAGAACAGAGCTTTAACACGGGAGG 23TGAGCGTCTGACCTCCCGTGTTAAAGCTCTGTTCTCCGTTCTGAACTACGAACGTGCTCGTCGTCCGGGTCTGCTGGGTGCTTCCGTTCTG 24BCGGTAACGTCAACTTTAACGAAGTACAGTTCCGGCGGCGGGTCCTGAGCACGAACACGCAGAACGAAGGTACGCCAAGCACGGTGGATGT 24TGGTCTGGACGACATCCACCGTGCTTGGCGTACCTTCGTTCTGCGTGTTCGTGCTCAGGACCCGCCGCCGGAACTGTACTTCGTTAAAGTT 25BCGTAACGACGAACGCAGTAGGTGTTCTGCGGTTTGATGATGGAAGCGATAACTTCGGTCAGACGGTCCTGCGGGATGGTGTCGTACGCGC 25TGACGTTACCGGCGCGTACGACACCATCCCGCAGGACCGTCTGACCGAAGTTATCGCTTCCATCATCAAACCGCAGAACACCTACTGCGTT 26BGACGCATGTACGGCTGCAGGTCGGTCAGGGTGGAAACGTGGGATTTGAATGCTTTACGAACGTGACCGTGAGCAGCTTTCTGAACAACAG 26TCGTCGTTACGCTGTTGTTCAGAAAGCTGCTCACGGTCACGTTCGTAAAGCATTCAAATCCCACGTTTCCACCCTGACCGACCTGCAGCCG 27BGACCGGAGGAAGCTTCGTTCAGGGAGGAGGACTGTTCGATAACAACAGCGTCACGCAGCGGGGAGGTTTCCTGCAGGTGAGCAACGAACT 27TTACATGCGTCAGTTCGTTGCTCACCTGCAGGAAACCTCCCCGCTGCGTGACGCTGTTGTTATCGAACAGTCCTCCTCCCTGAACGAAGCT 28BAACCCTGCGGGATACCCTGGCACTGAACGTAGGATTTACCACGGATACGAACAGCGTGGTGGCACATGAAACGCAGGAAAACGTCGAACA 28TTCCTCCGGTCTGTTCGACGTTTTCCTGCGTTTCATGTGCCACCACGCTGTTCGTATCCGTGGTAAATCCTACGTTCAGTGCCAGGGTATC 29BGCAGCAGCAGACCGTCACGACGGATACCAGCGAACAGTTTGTTTTCCATGTCACCGTAGCACAGGGAGCACAGCAGGGTGGACAGGATGG 29TCCGCAGGGTTCCATCCTGTCCACCCTGCTGTGCTCCCTGTGCTACGGTGACATGGAAAACAAACTGTTCGCTGGTATCCGTCGTGACGGT 30BCGTATTCCGGAACACCACGAACCAGGGTACGCAGGAAGGTTTTAGCGTGGGTCAGGTGCGGAGTAACCAGCAGGAAGTCGTCAACCAGAC 30TCTGCTGCTGCGTCTGGTTGACGACTTCCTGCTGGTTACTCCGCACCTGACCCACGCTAAAACCTTCCTGCGTACCCTGGTTCGTGGTGTT 31BGAGCCGGCATCTGAACGAAAGCGGTGCCACCCAGAGCTTCGTCTTCAACCGGGAAGTTAACAACGGTTTTACGCAGGTTTACAACGCAAC 31TCCGGAATACGGTTGCGTTGTAAACCTGCGTAAAACCGTTGTTAACTTCCCGGTTGAAGACGAAGCTCTGGGTGGCACCGCTTTCGTTCAG 32BGGATGGAGGTACGAGCGTAGGAGGAGTAGTCGGACTGAACTTCCAGGGTACGGGTGTCCAGCAGCAGACCGCACCACGGGAACAGACCGT 32TATGCCGGCTCACGGTCTGTTCCCGTGGTGCGGTCTGCTGCTGGACACCCGTACCCTGGAAGTTCAGTCCGACTACTCCTCCTACGCTCGT 33BGGGAGTGGCATTTCAGACGCAGAACACCGAACAGTTTACGACGCATGTTACGACCAGCTTTGAAACCACGGTTGAAGGTCAGGGAAGCAC 33TACCTCCATCCGTGCTTCCCTGACCTTCAACCGTGGTTTCAAAGCTGGTCGTAACATGCGTCGTAAACTGTTCGGTGTTCTGCGTCTGAAA 34BACGCGTGGAAACGGTAAGCCTGCAGCAGCAGGATTTTGTAGATGTTGGTGCAAACGGTCTGCAGGGAGTTTACCTGCAGGTCCAGGAACA 34TTGCCACTCCCTGTTCCTGGACCTGCAGGTAAACTCCCTGCAGACCGTTTGCACCAACATCTACAAAATCCTGCTGCTGCAGGCTTACCGT 35BAGTAGCACAGGGAAGCGGTGTCGGAGATAACACGCAGGAAGAAGGTCGGGTTTTTCCAAACCTGCTGGTGGAACGGCAGCTGCAGAACGC 35TTTCCACGCGTGCGTTCTGCAGCTGCCGTTCCACCAGCAGGTTTGGAAAAACCCGACCTTCTTCCTGCGTGTTATCTCCGACACCGCTTCC 36BGGCACAGCCACTGAACAGCTTCGGACGGCAGCGGACCAGCAGCACCTTTAGCACCCAGGGACATACCAGCGTTTTTAGCTTTCAGGATGG 36TCTGTGCTACTCCATCCTGAAAGCTAAAAACGCTGGTATGTCCCTGGGTGCTAAAGGTGCTGCTGGTCCGCTGCCGTCCGAAGCTGTTCAG 37BACAGCTGGGTCTGAGCGGTACGCAGGGAACCCAGCAGCGGAACGTAGGTAACACGGTGACGGGTCAGTTTCAGCAGGAAAGCCTGGT 37TTGGCTGTGCCACCAGGCTTTCCTGCTGAAACTGACCCGTCACCGTGTTACCTACGTTCCGCTGCTGGGTTCCCTGCGTACCGCTCAG 38BACGGCAGAGCCGGGTTAGCAGCAGCTTCCAGAGCGGTCAGGGTGGT ACCCGGCAGTTTACGGG 38TACCCAGCTGTCCCGTAAACTGCCGGGTACCACCCTGACCGCTCTGG AAGCTGCTGCTAACCCGG 39BGCGTGCCTCGAGGAATTCGGATCCATTAGTCCAGGATGGTTTTGAA GTCG 39TCTCTGCCGTCCGACTTCAAAACCATCCTGGACTAATGGATCCGAAT TCCTCGAGGCACGC

[0272] TABLE 10B (SEQ ID NO:721)GCACGCGGGAGCTCTAGAGTCGACCATATGCCGCGTGCTCCGCGTTGCCGTGCTGTTCGTTCCCTGCTGCGTTCCCACTATCGCGAAGTTCTGCCGCTGGCTACCTTCGTTCGTCGTCTGGGCCCGCAGGGTTGGCGTCTGGTTCAGCGTGGTGACCCGGCTGCTTTCCGTGCTCTGGTTGCTCAGTGCCTGGTTTGCGTTCCGTGGGACGCTCGTCCGCCGCCGGCTGCTCCGTCCTTCCGTCAGGTTTCCTGCCTGAAAGAACTGGTTGCTCGTGTTCTGCAGCGTCTGTGCGAACGTGGTGCTAAAAACGTTCTGGCTTTCGGTTTCGCTCTGCTGGACGGTGCTCGTGGTGGTCCGCCGGAAGCATTCACCACCTCCGTTCGTTCCTACCTGCCGAACACCGTTACCGACGCTCTGCGTGGTTCCGGTGCTTGGGGTCTGCTGCTGCGTCGTGTTGGTGACGACGTTCTGGTTCACCTGCTGGCTCGTTGCGCTCTGTTCGTTCTGGTTGCTCCGTCCTGCGCTTACCAGGTTTGTGGTCCGCCGCTGTACCAGCTGGGTGCTGCTACCCAGGCTCGTCCGCCGCCGCACGCTTCCGGTCCGCGTCGTCGTCTGGGTTGCGAACGTGCTTGGAACCACTCCGTTCGTGAAGCTGGTGTTCCGCTGGGTCTGCCGGCTCCGGGTGCTCGTCGTCGTGGTGGTTCCGCTTCCCGTTCCCTGCCGCTGCCGAAACGTCCGCGTCGTGGTGCTGCTCCGGAACCGGAACGTACCCCGGTTGGTCAGGGTTCCTGGGCTCACCCGGGTCGTACCCGTGGTCCGTCCGACCGTGGTTTCTGCGTTGTTTCCCCGGCTCGTCCGGCTGAAGAAGCTACCTCCCTGGAAGGTGCTCTGTCCGGCACCCGTCACTCCCACCCGTCCGTTGGTCGTCAGCACCACGCTGGTCCGCCGTCCACCTCCCGTCCGCCGCGTCCGTGGGACACCCCGTGCCCGCCGGTTTACGCTGAAACCAAACACTTCCTGTACTCCTCCGGTGACAAAGAACAGCTGCGTCCGTCCTTCCTGCTGTCCTCCCTGCGTCCGTCCCTGACCGGTGCTCGTCGTCTGGTTGAAACTATCTTCCTGGGTTCCCGTCCGTGGATGCCGGGCACCCCGCGTCGTCTGCCGCGTCTGCCGCAGCGTTACTGGCAGATGCGTCCGCTGTTCCTGGAACTGCTGGGCAACCACGCTCAGTGCCCGTATGGTGTTCTGCTGAAAACCCACTGCCCGCTGCGTGCTGCTGTTACCCCGGCTGCTGGTGTTTGCGCTCGTGAAAAACCGCAGGGTTCCGTTGCTGCTCCGGAAGAAGAAGATACCGACCCGCGTCGTCTGGTTCAGCTGCTGCGTCAGCACTCCTCCCCGTGGCAGGTTTACGGTTTCGTTCGTGCATGCCTGCGTCGTCTGGTTCCGCCGGGTCTGTGGGGTTCCCGTCACAACGAACGTCGTTTCCTGCGTAACACCAAAAAATTCATCTCCCTGGGTAAACACGCTAAACTGTCCCTGCAGGAACTGACCTGGAAAATGTCCGTTCGTGACTGCGCTTGGCTGCGTCGTTCTCCGGGTGTTGGTTGCGTTCCGGCTGCTGAACACCGTCTGCGTGAAGAAATCCTGGCTAAATTCCTGCACTGGCTGATGTCCGTATACGTTGTTGAACTGCTGCGTTCCTTCTTCTACGTTACCGAAACCACCTTCCAGAAAAACCGTCTGTTCTTCTACCGTAAATCCGTTTGGTCCAAACTGCAGTCCATCGGTATCCGTCAGCACCTGAAACGTGTTCAGCTGCGTGAACTGTCCGAAGCTGAAGTTCGTCAGCACCGTGAAGCTCGTCCGGCTCTGCTGACCTCCCGTCTGCGTTTCATCCCGAAACCGGACGGTCTGCGTCCGATCGTAAACATGGACTACGTTGTTGGTGCTCGTACCTTCCGTCGTGAAAAACGTGCTGAGCGTCTGACCTCCCGTGTTAAAGCTCTGTTCTCCGTTCTGAACTACGAACGTGCTCGTCGTCCGGGTCTGCTGGGTGCTTCCGTTCTGGGTCTGGACGACATCCACCGTGCTTGGCGTACCTTCGTTCTGCGTGTTCGTGCTCAGGACCCGCCGCCGGAACTGTACTTCGTTAAAGTTGACGTTACCGGCGCGTACGACACCATCCCGCAGGACCGTCTGACCGAAGTTATCGCTTCCATCATCAAACCGCAGAACACCTACTGCGTTCGTCGTTACGCTGTTGTTCAGAAAGCTGCTCACGGTCACGTTCGTAAAGCATTCAAATCCCACGTTTCCACCCTGACCGACCTGCAGCCGTACATGCGTCAGTTCGTTGCTCACCTGCAGGAAACCTCCCCGCTGCGTGACGCTGTTGTTATCGAACAGTCCTCCTCCCTGAACGAAGCTTCCTCCGGTCTGTTCGACGTTTTCCTGCGTTTCATGTGCCACCACGCTGTTCGTATCCGTGGTAAATCCTACGTTCAGTGCCAGGGTATCCCGCAGGGTTCCATCCTGTCCACCCTGCTGTGCTCCCTGTGCTACGGTGACATGGAAAACAAACTGTTCGCTGGTATCCGTCGTGACGGTCTGCTGCTGCGTCTGGTTGACGACTTCCTGCTGGTTACTCCGCACCTGACCCACGCTAAAACCTTCCTGCGTACCCTGGTTCGTGGTGTTCCGGAATACGGTTGCGTTGTAAACCTGCGTAAAACCGTTGTTAACTTCCCGGTTGAAGACGAAGCTCTGGGTGGCACCGCTTTCGTTCAGATGCCGGCTCACGGTCTGTTCCCGTGGTGCGGTCTGCTGCTGGACACCCGTACCCTGGAAGTTCAGTCCGACTACTCCTCCTACGCTCGTACCTCCATCCGTGCTTCCCTGACCTTCAACCGTGGTTTCAAAGCTGGTCGTAACATGCGTCGTAAACTGTTCGGTGTTCTGCGTCTGAAATGCCACTCCCTGTTCCTGGACCTGCAGGTAAACTCCCTGCAGACCGTTTGCACCAACATCTACAAAATCCTGCTGCTGCAGGCTTACCGTTTCCACGCGTGCGTTCTGCAGCTGCCGTTCCACCAGCAGGTTTGGAAAAACCCGACCTTCTTCCTGCGTGTTATCTCCGACACCGCTTCCCTGTGCTACTCCATCCTGAAAGCTAAAAACGCTGGTATGTCCCTGGGTGCTAAAGGTGCTGCTGGTCCGCTGCCGTCCGAAGCTGTTCAGTGGCTGTGCCACCAGGCTTTCCTGCTGAAACTGACCCGTCACCGTGTTACCTACGTTCCGCTGCTGGGTTCCCTGCGTACCGCTCAGACCCAGCTGTCCCGTAAACTGCCGGGTACCACCCTGACCGCTCTGGAAGCTGCTGCTAACCCGGCTCTGCCGTCCGACTTCAAAACCATCCTGGACTAATGGATCCGAATTCCTCGAGGCACG C

[0273] The present invention also provides transgenic animals (i.e.,mammals transgenic for a human or other TRT gene sequence) expressing anhTRT or other TRT polynucleotide or polypeptide. In one embodiment, hTRTis secreted into the milk of a transgenic mammal such as a transgenicbovine, goat, or rabbit. Methods for production of such animals arefound, e.g., in Heyneker et al., PCT WO 91/08216.

[0274] The hTRT proteins and complexes of the invention, including thosemade using the expression systems disclosed herein supra, may bepurified using a variety of general methods known in the art inaccordance with the specific methods provided by the present invention(e.g., infra). One of skill in the art will recognize that afterchemical synthesis, biological expression, or purification, the hTRTprotein may possess a conformation different than a native conformationof naturally occurring telomerase. In some instances, it may be helpfulor even necessary to denature (e.g., including reduction of disulfide orother linkages) the polypeptide and then to cause the polypeptide tore-fold into the preferred conformation. Productive refolding may alsorequire the presence of hTR (or hTR fragments). Methods of reducing anddenaturing proteins and inducing re-folding are well known to those ofskill in the art (see, e.g., Debinski et al., 1993, J. Biol. Chem.,268:14065; Kreitman and Pastan, 1993, Bioconjug. Chem., 4:581; andBuchner et al., 1992, Anal. Biochem., 205:263; and McCaman et al., 1985,J. Biotech. 2:177). See also PCT Publication WO 96/40868, supra.

[0275] D) Complexes of Human TRT and Human Telomerase RNA,Telomerase-Associated Proteins, and Other Biomolecules Produced byCoexpression and Other Means

[0276] hTRT polypeptides of the invention can associate in vivo and invitro with other biomolecules, including RNAs (e.g., hTR), proteins(e.g., telomerase-associated proteins), DNA (e.g., telomeric DNA,[T₂AG₃]_(N)), and nucleotides, such as (deoxy)ribonucleotidetriphosphates. These associations can be exploited to assay hTRTpresence or function, to identify or purify hTRT ortelomerase-associated molecules, and to analyze hTRT or telomerasestructure or function in accordance with the methods of the presentinvention.

[0277] In one embodiment, the present invention provides hTRT complexedwith (e.g., associated with or bound to) a nucleic acid, usually an RNA,for example to produce a telomerase holoenzyme. In one embodiment, thebound RNA is capable of acting as a template for telomerase-mediated DNAsynthesis. Examples of RNAs that may be complexed with the hTRTpolypeptide include a naturally occurring host cell telomerase RNA, ahuman telomerase RNA (e.g., hTR; U.S. Pat. No. 5,583,016), an hTRsubsequence or domain, a synthetic RNA, or other RNAs. The RNA-hTRTprotein complex (an RNP) typically exhibits one or more telomeraseactivities, such as telomerase catalytic activities. These hTRT-hTR RNPs(or other hTRT-RNA complexes) can be produced by a variety of methods,as described infra for illustrative purposes, including in vitroreconstitution, by co-expression of hTRT and hTR (or other RNA) in vitro(i.e., in a cell free system), in vivo reconstitution, or ex vivoreconstitution.

[0278] Thus, the present invention provides, in one embodiment, anhTRT-hTR complex (or other hTRT-RNA complex) formed in vitro by mixingseparately purified components (“in vitro reconstitution;” see, e.g.,U.S. Pat. No. 5,583,016 for a description of reconstitution; also seeAutexier et al., EMBO J. 15:5928). In one embodiment the hTRT protein isproduced by recombinant expression in human or non-human cells, e.g., asdescribed supra, and subsequently purified using protein purificationmethods (e.g., chromatography, affinity purification). In a particularembodiment, the recombinant hTRT protein is purified to homogeneity. Thepurified hTRT protein is combined with separately purified hTR, whichmay be produced using an in vitro transcription system, by chemicalsynthesis, or by other methods and purified using standard RNApurification techniques (see Melton et al., 1984, Nucl. Acids Res.12:7035; Studier et al., 1986, J. Mol. Biol. 189:113).

[0279] In an alternative embodiment, the invention provides telomeraseRNPs produced by coexpression of the hTRT polypeptide and an RNA (e.g.,hTR) in vitro in a cell-free transcription-translation system (e.g.wheat germ or rabbit reticulocyte lysate). As shown in Example 7, invitro co-expression of a recombinant hTRT polypeptide and hTR results inproduction of telomerase catalytic activity (as measured by a TRAPassay).

[0280] Further provided by the present invention are telomerase RNPsproduced by expression of the hTRT polypeptide in a cell, e.g., amammalian cell, in which hTR is naturally expressed or in which hTR (oranother RNA capable of forming a complex with the hTRT protein) isintroduced or expressed by recombinant means. Thus, in one embodiment,hTRT is expressed in a telomerase negative human cell in which hTR ispresent (e.g., BJ or IMP90 cells), allowing the two molecules toassemble into an RNP. In another embodiment, hTRT is expressed in ahuman or non-human cell in which hTR is recombinantly expressed. Methodsfor expression of hTR in a cell are found in U.S. Pat. No. 5,583,016.Further, a clone containing a cDNA encoding the RNA component oftelomerase has been placed on deposit as pGRN33 (ATCC 75926). Genomicsequences encoding the RNA component of human telomerase are also ondeposit in the ˜15 kb SauIIIA1 to HindIII insert of lambda clone 28-1(ATCC 75925). For expression in eukaryotic cells the hTRT sequence willtypically be operably linked to a transcription initiation sequence (RNApolymerase binding site) and transcription terminator sequences (see,e.g., PCT Publication WO 96/01835; Feng et al., 1995, Science 269:1236).

[0281] The present invention further provides recombinantly produced orsubstantially purified hTRT polypeptides coexpressed and/or associatedwith so-called “telomerase-associated proteins.” Thus, the presentinvention provides hTRT coexpressed with, or complexed with, otherproteins (e.g., telomerase-associated proteins). Telomerase-associatedproteins are those proteins that copurify with human telomerase and/orthat may play a role in modulating telomerase function or activity, forexample by participating in the association of telomerase with telomericDNA. Examples of telomerase-associated proteins include (but are notlimited to) the following proteins and/or their human homologs:nucleolin (see, Srivastava et al., 1989, FEBS Letts. 250:99); EF2H(elongation factor 2 homolog; see Nomura et al. 1994, DNA Res. (Japan)1:27, GENBANK accession #D21163); TP1/TLP1 (Harrington et al., 1997,Science 275:973; Nakayama, 1997, Cell 88:875); the human homologue ofthe Tetrahymena p95 or p95 itself (Collins et al., 1995, Cell 81:677);TPC2 (a telomere length regulatory protein; ATCC accession number 97708;TPC3 (also a telomere length regulatory protein; ATCC accession number97707; DNA-binding protein B (dbpB; Horwitz et al., 1994, J. Biol. Chem.269:14130; and Telomere Repeat Binding Factors (TRF 1 & 2; Chang et al.,1995, Science 270:1663; Chong et al., 1997, Hum Mol Genet 6:69); EST1, 3and 4 (Lendvay et al., 1996, Genetics 144:1399, Nugent et al., 1996,Science 274:249, Lundblad et al., 1989, Cell 57:633); and End-cappingfactor (Cardenas et al., 1993, Genes Dev. 7:883).

[0282] Telomerase associated proteins can be identified on the basis ofco-purification with, or binding to, hTRT protein or the hTRT-hTR RNP.Alternatively, they can be identified on the basis of binding to an hTRTfusion protein, e.g., a GST-hTRT fusion protein or the like, asdetermined by affinity purification (see, Ausubel et al. Ch 20). Aparticularly useful technique for assessing protein-proteininteractions, which is applicable to identifying hTRT-associatedproteins, is the two hybrid screen method of Chien et al. (Proc. Natl.Acad. Sci. USA 88:9578 [1991]; see also Ausubel et al., supra, at Ch.20). This screen identifies protein-protein interactions in vivo throughreconstitution of a transcriptional activator, the yeast Gal4transcription protein (see, Fields and Song, 1989, Nature 340:245). Themethod is based on the properties of the yeast Gal4 protein, whichconsists of separable domains responsible for DNA-binding andtranscriptional activation. Polynucleotides, usually expression vectors,encoding two hybrid proteins are constructed. One polynucleotidecomprises the yeast Gal4 DNA-binding domain fused to a polypeptidesequence of a protein to be tested for an hTRT interaction (e.g.,nucleolin or EF2H). Alternatively the yeast Gal4 DNA-binding domain isfused to cDNAs from a human cell, thus creating a library of humanproteins fused to the Gal4 DNA binding domain for screening fortelomerase associated proteins. The other polynucleotide comprises theGal4 activation domain fused to an hTRT polypeptide sequence. Theconstructs are introduced into a yeast host cell. Upon expression,intermolecular binding between hTRT and the test protein canreconstitute the Gal4 DNA-binding domain with the Gal4 activationdomain. This leads to the transcriptional activation of a reporter gene(e.g., lacZ, HIS3) operably linked to a Gal4 binding site. By selectingfor, or by assaying the reporter, gene colonies of cells that contain anhTRT interacting protein or telomerase associated protein can beidentified. Those of skill will appreciate that there are numerousvariations of the 2-hybrid screen, e.g., the LexA system (Bartel et al,1993, in Cellular Interactions in Development: A Practical Approach Ed.Hartley, D. A. (Oxford Univ. Press) pp. 153-79).

[0283] Another useful method for identifying telomerase-associatedproteins is a three-hybrid system (see, e.g., Zhang et al., 1996, Anal.Biochem. 242:68; Licitra et al., 1996, Proc. Natl. Acad. Sci. USA93:12817). The telomerase RNA component can be utilized in this systemwith the TRT or hTRT protein and a test protein. Another useful methodfor identifying interacting proteins, particularly (i.e., proteins thatheterodimerize or form higher order heteromultimers), is the E.coli/BCCP interactive screening system (see, Germino et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:933; Guarente (1993) Proc. Natl. Acad. Sci.(U.S.A.) 90:1639).

[0284] The present invention also provides complexes of telomere bindingproteins (which may or may not be telomerase associated proteins) andhTRT (which may or may not be complexed with hTR, other RNAs, or one ormore telomerase associated proteins). Examples of telomere bindingproteins include TRF1 and TRF2 (supra); rnpA1, rnpA2, RAP1 (Buchman etal., 1988, Mol. Cell. Biol. 8:210, Buchman et al., 1988, Mol. Cell.Biol. 8:5086), SIR3 and SIR4 (Aparicio et al, 1991, Cell 66:1279), TEL1(Greenwell et al., 1995, Cell 82:823; Morrow et al., 1995, Cell 82:831);ATM (Savitsky et al., 1995, Science 268:1749), end-capping factor(Cardenas et al., 1993, Genes Dev. 7:883), and corresponding humanhomologs. The aforementioned complexes may be produced generally asdescribed supra for complexes of hTRT and hTR or telomerase associatedproteins, e.g., by mixing or co-expression in vitro or in vivo.

[0285] V. Antibodies and Other Binding Agents

[0286] In a related aspect, the present invention provides antibodiesthat are specifically immunoreactive with hTRT, including polyclonal andmonoclonal antibodies, antibody fragments, single chain antibodies,human and chimeric antibodies, including antibodies or antibodyfragments fused to phage coat or cell surface proteins, and others knownin the art and described herein. The antibodies of the invention canspecifically recognize and bind polypeptides that have an amino acidsequence that is substantially identical to the amino acid sequence setforth in FIG. 17 SEQ ID NO:2, or an immunogenic fragment thereof orepitope on the protein defined thereby. The antibodies of the inventioncan exhibit a specific binding affinity for hTRT of at least about 10⁷,10⁸, 10⁹, or 10¹⁰ M⁻¹, and may be polyclonal, monoclonal, recombinant orotherwise produced. The invention also provides anti-hTRT antibodiesthat recognize an hTRT conformational epitope (e.g., an epitope on thesurface of the hTRT protein or a telomerase RNP). Likely conformationalepitopes can be identified, if desired, by computer-assisted analysis ofthe hTRT protein sequence, comparison to the conformation of relatedreverse transcriptases, such as the p66 subunit of HIV-1 (see, e.g.,FIG. 3), or empirically. Anti-hTRT antibodies that recognizeconformational epitopes have utility, inter alia, in detection andpurification of human telomerase and in the diagnosis and treatment ofhuman disease.

[0287] For the production of anti-hTRT antibodies, hosts such as goats,sheep, cows, guinea pigs, rabbits, rats, or mice, may be immunized byinjection with hTRT protein or any portion, fragment or oligopeptidethereof which retains immunogenic properties. In selecting hTRTpolypeptides for antibody induction, one need not retain biologicalactivity; however, the protein fragment, or oligopeptide must beimmunogenic, and preferably antigenic. Immunogenicity can be determinedby injecting a polypeptide and adjuvant into an animal (e.g., a rabbit)and assaying for the appearance of antibodies directed against theinjected polypeptide (see, e.g., Harlow and Lane, ANTIBODIES: ALABORATORY MANUAL, COLD SPRING HARBOR LABORATORY, New York (1988), whichis incorporated in its entirety and for all purposes, e.g., at Chapter5). Peptides used to induce specific antibodies typically have an aminoacid sequence consisting of at least five amino acids, preferably atleast 8 amino acids, more preferably at least 10 amino acids. Usuallythey will mimic or have substantial sequence identity to all or acontiguous portion of the amino acid sequence of the protein of SEQ IDNO:2. Short stretches of hTRT protein amino acids may be fused withthose of another protein, such as keyhole limpet hemocyanin, and ananti-hTRT antibody produced against the chimeric molecule. Depending onthe host species, various adjuvants may be used to increaseimmunological response.

[0288] The antigen is presented to the immune system in a fashiondetermined by methods appropriate for the animal. These and otherparameters are generally well known to immunologists. Typically,injections are given in the footpads, intramuscularly, intradermally,perilymph nodally or intraperitoneally. The immunoglobulins produced bythe host can be precipitated, isolated and purified by routine methods,including affinity purification.

[0289] Illustrative examples of immunogenic hTRT peptides include areprovided in Example 8. In addition, Example 8 describes the productionand use of anti-hTRT polyclonal antibodies.

[0290] A) Monoclonal Antibodies

[0291] Monoclonal antibodies to hTRT proteins and peptides may beprepared in accordance with the methods of the invention using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Koehler and Milstein(Nature 256:495 [1975]), the human B-cell hybridoma technique (Kosbor etal., 1983, Immunol. Today 4:72; Cote et al., 1983, Proc. Natl. Acad.Sci. USA, 80:2026), and the EBV-hybridoma technique (Cole et al.,MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Liss Inc, New YorkN.Y., pp 77-96 [1985]).

[0292] In one embodiment, appropriate animals are selected and theappropriate immunization protocol followed. The production of non-humanmonoclonal antibodies, e.g., murine, lagomorpha, equine, is well knownand can be accomplished by, for example, immunizing an animal with apreparation containing hTRT or fragments thereof. In one method, afterthe appropriate period of time, the spleens of the animals are excisedand individual spleen cells are fused, typically, to immortalizedmyeloma cells under appropriate selection conditions. Thereafter, thecells are clonally separated and the supernatants of each clone (e.g.,hybridoma) are tested for the production of an appropriate antibodyspecific for the desired region of the antigen. Techniques for producingantibodies are well known in the art. See, e.g., Goding et al.,MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2D ED.) Acad. Press,N.Y., and Harlow and Lane, supra, each of which is incorporated in itsentirety and for all purposes. Other suitable techniques involve the invitro exposure of lymphocytes to the antigenic polypeptides oralternatively, to selection of libraries of antibodies in phage orsimilar vectors (see, infra).

[0293] B) Human Antibodies

[0294] In another aspect of the invention, human antibodies against anhTRT polypeptide are provided. Human monoclonal antibodies against aknown antigen can also be made using transgenic animals having elementsof a human immune system (see, e.g., U.S. Pat. Nos. 5,569,825 and5,545,806, both of which are incorporated by reference in their entiretyfor all purposes) or using human peripheral blood cells (Casali et al.,1986, Science 234:476). Some human antibodies are selected bycompetitive binding experiments, or otherwise, to have the same epitopespecificity as a particular mouse antibody.

[0295] In an alternative embodiment, human antibodies to an hTRTpolypeptide can be produced by screening a DNA library from human Bcells according to the general protocol outlined by Huse et al., 1989,Science 246:1275, which is incorporated by reference. Antibodies bindingto the hTRT polypeptide are selected. Sequences encoding such antibodies(or binding fragments) are then cloned and amplified. The protocoldescribed by Huse is often used with phage-display technology.

[0296] C) Humanized or Chimeric Antibodies

[0297] The invention also provides anti-hTRT antibodies that are madechimeric, human-like or humanized, to reduce their potentialantigenicity, without reducing their affinity for their target.Preparation of chimeric, human-like and humanized antibodies have beendescribed in the art (see, e.g., U.S. Pat. Nos. 5,585,089 and 5,530,101;Queen, et al., 1989, Proc. Nat'l Acad. Sci. USA 86:10029; and Verhoeyanet al., 1988, Science 239:1534; each of which is incorporated byreference in their entirety and for all purposes). Humanizedimmunoglobulins have variable framework regions substantially from ahuman immunoglobulin (termed an acceptor immunoglobulin) andcomplementarity determining regions substantially from a non-human(e.g., mouse) immunoglobulin (referred to as the donor immunoglobulin).The constant region(s), if present, are also substantially from a humanimmunoglobulin.

[0298] In some applications, such as administration to human patients,the humanized (as well as human) anti-hTRT antibodies of the presentinvention offer several advantages over antibodies from murine or otherspecies: (1) the human immune system should not recognize the frameworkor constant region of the humanized antibody as foreign, and thereforethe antibody response against such an injected antibody should be lessthan against a totally foreign mouse antibody or a partially foreignchimeric antibody; (2) because the effector portion of the humanizedantibody is human, it may interact better with other parts of the humanimmune system; and (3) injected humanized antibodies have a half-lifeessentially equivalent to naturally occurring human antibodies, allowingsmaller and less frequent doses than antibodies of other species. Asimplicit from the foregoing, anti hTRT antibodies have application inthe treatment of disease, i.e., to target telomerase-positive cells.

[0299] D) Phage Display

[0300] The present invention also provides anti-hTRT antibodies (orbinding compositions) produced by phage display methods (see, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047; andVaughan et al., 1996, Nature Biotechnology, 14: 309; each of which isincorporated by reference in its entirety for all purposes). In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outer surfaces. Antibodies are usuallydisplayed as Fv or Fab fragments. Phage displaying antibodies with adesired specificity are selected by affinity enrichment to an hTRTpolypeptide.

[0301] In a variation of the phage-display method, humanized antibodieshaving the binding specificity of a selected murine antibody can beproduced. In this method, either the heavy or light chain variableregion of the selected murine antibody is used as a starting material.If, for example, a light chain variable region is selected as thestarting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding for thehTRT polypeptide (e.g., at least 10⁸ and preferably at least 10⁹ M⁻¹) isselected. The human heavy chain variable region from this phage thenserves as a starting material for constructing a further phage library.In this library, each phage displays the same heavy chain variableregion (i.e., the region identified from the first display library) anda different light chain variable region. The light chain variableregions are obtained from a library of rearranged human variable lightchain regions. Again, phage showing strong specific binding areselected. These phage display the variable regions of completely humananti-hTRT antibodies. These antibodies usually have the same or similarepitope specificity as the murine starting material.

[0302] E) Hybrid Antibodies

[0303] The invention also provides hybrid antibodies that share thespecificity of antibodies against an hTRT polypeptide but are alsocapable of specific binding to a second moiety. In such hybridantibodies, one heavy and light chain pair is usually from an anti-hTRTantibody and the other pair from an antibody raised against anotherepitope or protein. This results in the property of multi-functionalvalency, i.e., ability to bind at least two different epitopessimultaneously, where at least one epitope is the epitope to which theanti-complex antibody binds. Such hybrids can be formed by fusion ofhybridomas producing the respective component antibodies, or byrecombinant techniques. Such hybrids can be used to carry a compound(i.e., drug) to a telomerase-positive cell (i.e., a cytotoxic agent isdelivered to a cancer cell).

[0304] Immunoglobulins of the present invention can also be fused tofunctional regions from other genes (e.g., enzymes) to produce fusionproteins (e.g., immunotoxins) having useful properties.

[0305] F) Anti-Idiotypic Antibodies

[0306] Also useful are anti-idiotype antibodies which can be isolated bythe above procedures. Anti-idiotypic antibodies may be prepared by, forexample, immunization of an animal with the primary antibody (i.e.,anti-hTRT antibodies or hTRT-binding fragments thereof). For anti-hTRTantibodies, anti-idiotype antibodies whose binding to the primaryantibody is inhibited by an hTRT polypeptide or fragments thereof areselected. Because both the anti-idiotypic antibody and the hTRTpolypeptide or fragments thereof bind the primary immunoglobulin, theanti-idiotypic immunoglobulin can represent the “internal image” of anepitope and thus can substitute for the hTRT polypeptide in assays orcan be used to bind (i.e., inactivate) anti-hTRT antibodies, e.g., in apatient. Anti-idiotype antibodies can also interact with telomeraseassociated proteins. Administration of such antibodies can affecttelomerase function by titrating out or competing with hTRT in bindingto hTRT-associated proteins.

[0307] G) General

[0308] The antibodies of the invention may be of any isotype, e.g., IgM,IgD, IgG, IgA, and IgE, with IgG, IgA and IgM often preferred. Humanizedantibodies may comprise sequences from more than one class or isotype.

[0309] In another embodiment of the invention, fragments of the intactantibodies described above are provided. Typically, these fragments cancompete with the intact antibody from which they were derived forspecific binding to the hTRT polypeptide, and bind with an affinity ofat least 10⁷, 10⁸, 10⁹ M⁻¹, or 10¹⁰ M⁻¹. Antibody fragments includeseparate heavy chains, light chains, Fab, Fab′ F(ab′)₂, Fabc, and Fv.Fragments can be produced by enzymatic or chemical separation of intactimmunoglobulins. For example, a F(ab′)₂ fragment can be obtained from anIgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 usingstandard methods such as those described in Harlow and Lane, supra. Fabfragments may be obtained from F(ab′)₂ fragments by limited reduction,or from whole antibody by digestion with papain in the presence ofreducing agents (see generally, Paul, W., ed FUNDAMENTAL IMMUNOLOGY2^(ND) Raven Press, N.Y., 1989, Ch. 7, incorporated by reference in itsentirety for all purposes). Fragments can also be produced byrecombinant DNA techniques. Segments of nucleic acids encoding selectedfragments are produced by digestion of full-length coding sequences withrestriction enzymes, or by de novo synthesis. Often fragments areexpressed in the form of phage-coat fusion proteins.

[0310] Many of the immunoglobulins described above can undergonon-critical amino-acid substitutions, additions or deletions in boththe variable and constant regions without loss of binding specificity oreffector functions, or intolerable reduction of binding affinity (i.e.,below about 10⁷ M⁻¹). Usually, immunoglobulins incorporating suchalterations exhibit substantial sequence identity to a referenceimmunoglobulin from which they were derived. A mutated immunoglobulincan be selected having the same specificity and increased affinitycompared with a reference immunoglobulin from which it was derived.Phage-display technology offers useful techniques for selecting suchimmunoglobulins. See, e.g., Dower et al., WO 91/17271 McCafferty et al.,WO 92/01047; and Huse, WO 92/06204.

[0311] The antibodies of the present invention can be used with orwithout modification. Frequently, the antibodies will be labeled byjoining, either covalently or non-covalently, a detectable label. Aslabeled binding entities, the antibodies of the invention areparticularly useful in diagnostic applications.

[0312] The anti-hTRT antibodies of the invention can be purified usingwell known methods. The whole antibodies, their dimers, individual lightand heavy chains, or other immunoglobulin forms of the present inventioncan be purified using the methods and reagents of the present inventionin accordance with standard procedures of the art, including ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see generally Scopes, PROTEINPURIFICATION: PRINCIPLES AND PRACTICE 3^(RD) EDITION (Springer-Verlag,N.Y., 1994)). Substantially pure immunoglobulins of at least about 90 to95%, or even 98 to 99% or more homogeneity are preferred.

[0313] VI. Purification of Human Telomerase

[0314] The present invention provides isolated human telomerase ofunprecedented purity. In particular, the present invention provides:purified hTRT of recombinant or nonrecombinant origin; purified hTRT-hTRcomplexes (i.e., RNPS) of recombinant, nonrecombinant, or mixed origin,optionally comprising one or more telomerase-associated proteins;purified naturally occurring human telomerase; and the like. Moreover,the invention provides methods and reagents for partially, substantiallyor highly purifying the above-molecules and complexes, includingvariants, fusion proteins, naturally occurring proteins, and the like(collectively referred to as “hTRT and/or hTRT complexes”).

[0315] Prior to the present disclosure, attempts had been made to purifythe telomerase enzyme complex to homogeneity had met with limitedsuccess. The methods provided in the aforelisted applications providepurification of telomerase by approximately up to 60,000-fold or morecompared to crude cell extracts. The present invention provides hTRT andhTRT complexes of even greater purity, in part by virtue of the novelimmunoaffinity reagents (e.g., anti-hTRT antibodies) of the presentinvention, and/or the reagents, cells, and methods provided herein forrecombinant expression of hTRT. Recombinant expression of hTRT and hTRTcomplexes facilitates purification because the desired molecules can beproduced at much higher levels than found in most expressing cellsoccurring in nature, and/or because the recombinant hTRT molecule can bemodified (e.g., by fusion with an epitope tag) such that it may beeasily purified.

[0316] It will be recognized that naturally occurring telomerase can bepurified from any telomerase-positive cell, and recombinant hTRT andhTRT complexes can be expressed and purified, inter alia, using any ofthe in vitro, in vivo, ex vivo, or plant or animal expression systemsdisclosed supra, or others/systems known in the art.

[0317] In one embodiment, the hTRT, telomerase and other compositions ofthe invention are purified using an immunoaffinity step, alone or incombination with other purification steps. Typically, an immobilized orimmobilizable anti-hTRT antibody, as provided by the present invention,is contacted with a sample, such as a cell lysate, that contains thedesired hTRT or hTRT-containing complex under conditions in whichanti-hTRT antibody binds the hTRT antigen. After removal of the unboundcomponents of the sample by methods well known in the art, the hTRTcomposition may be eluted, if desired, from the antibody, insubstantially pure form. In one embodiment, immunoaffinitychromatography methods well known in the art are used (see, e.g., Harlowand Lane, supra; and Ausubel, supra; Hermansan et al., 1992, IMMOBILIZEDAFFINITY LIGAND TECHNIQUES (Academic Press, San Diego)) in accordancewith the methods of the invention. In another illustrative embodiment,immunoprecipitation of anti-hTRT-immunoglobulin-hTRT complexes iscarried out using immobilized Protein A. Numerous variations andalternative immunoaffinity purification protocols suitable for use inaccordance with the methods and reagents of the invention are well-knownto those of skill.

[0318] In another embodiment, recombinant hTRT proteins can, as aconsequence of their high level of expression, be purified using routineprotein purification methods, such as ammonium sulfate precipitation,affinity columns (e.g., immunoaffinity), size-exclusion, anion andcation exchange chromatography, gel electrophoresis and the like (see,generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, N.Y. (1982)and Deutscher, METHODS IN ENZYMOLOGY VOL. 182: GUIDE TO PROTEINPURIFICATION, Academic Press, Inc. N.Y. (1990)) instead of, or inaddition to, immunoaffinity methods. Cation exchange methods can beparticularly useful due to the basic pI of the hTRT protein. Forexample, immobilized phosphate may be used as a cation exchangefunctional group (e.g., P-11 Phosphocellulose, Whatman catalog #4071 orCellulose Phosphate, Sigma catalog #C 3145). Immobilized phosphate hastwo advantageous features for hTRT purification—it is a cation exchangeresin, and it shows physical resemblance to the phosphate backbone ofnucleic acid. This can allow for affinity chromatography because hTRTbinds hTR and telomeric DNA. Other non-specific and specific nucleicacid affinity chromatography methods are also useful for purification(e.g., Alberts et al., 1971, Methods Enzymol. 21:198; Arnt-Jovin et al.,1975, Eur. J. Biochem. 54:411; Pharmacia catalog #27-5575-O₂). Furtherexploitation of this binding function of hTRT could include the use ofspecific nucleic acid (e.g., telomerase primer or hTR) affinitychromatography for purification (Chodosh et al., 1986, Mol. Cell. Biol.6:4723; Wu et al., 1987, Science 238:1247; Kadonaga, 1991, MethodsEnzymol. 208:10); immobilized Cibricon Blue Dye, which shows physicalresemblance to nucleotides, is another useful resin for hTRTpurification (Pharmacia catalog #17-0948-01 or Sigma catalog #C 1285),due to hTRT binding of nucleotides (e.g., as substrates for DNAsynthesis).

[0319] In one embodiment, hTRT proteins are isolated directly from an invitro or in vivo expression system in which other telomerase componentsare not coexpressed. It will be recognized that isolated hTRT proteinmay also be readily obtained from purified human telomerase or hTRTcomplexes, for example, by disrupting the telomerase RNP (e.g., byexposure to a mild or other denaturant) and separating the RNPcomponents (e.g., by routine means such as chromatography orimmunoaffinity chromatography).

[0320] Telomerase purification may be monitored using a telomeraseactivity assay (e.g., the TRAP assay, conventional assay, orprimer-binding assay), by measuring the enrichment of hTRT (e.g., byELISA), by measuring the enrichment of hTR, or other methods known inthe art.

[0321] The purified human telomerase, hTRT proteins, and hTRT complexesprovided by the present invention are, in one embodiment, highlypurified (i.e., at least about 90% homogeneous, more often at leastabout 95% homogeneous). Homogeneity can be determined by standard meanssuch as SDS-polyacrylamide gel electrophoresis and other means known inthe art (see, e.g., Ausubel et al, supra). It will be understood that,although highly purified human telomerase, hTRT protein, or hTRTcomplexes are sometimes desired, substantially purified (e.g., at leastabout 75% homogeneous) or partially purified (e.g., at least about 20%homogeneous) human telomerase, hTRT protein, or hTRT complexes areuseful in many applications, and are also provided by the presentinvention. For example, partially purified telomerase is useful forscreening test compounds for telomerase modulatory activity, and otheruses (see, infra and supra; see U.S. Pat. No. 5,645,986).

[0322] VII. Treatment of Telomerase-Related Disease

[0323] A) Introduction

[0324] The present invention provides hTRT polynucleotides,polypeptides, and antibodies useful for the treatment of human diseasesand disease conditions. The recombinant and synthetic hTRT gene products(protein and mRNA) of the invention can be used to create or elevatetelomerase activity in a cell, as well as to inhibit telomerase activityin cells in which it is not desired. Thus, inhibiting, activating orotherwise altering a telomerase activity (e.g., telomerase catalyticactivity, fidelity, processivity, telomere binding, etc.) in a cell canbe used to change the proliferative capacity of the cell. For example,reduction of telomerase activity in an immortal cell, such as amalignant tumor cell, can render the cell mortal. Conversely, increasingthe telomerase activity in a mortal cell (e.g., most human somaticcells) can increase the proliferative capacity of the cell. For example,expression of hTRT protein in dermal fibroblasts, thereby increasingtelomere length, will result in increased fibroblast proliferativecapacity; such expression can slow or reverse the age-dependent slowingof wound closure (see, e.g., West, 1994, Arch. Derm. 130:87).

[0325] Thus, in one aspect, the present invention provides reagents andmethods useful for treating diseases and conditions characterized by thepresence, absence, or amount of human telomerase activity in a cell andthat are susceptible to treatment using the compositions and methodsdisclosed herein. These diseases include, as described more fully below,cancers, other diseases of cell proliferation (particularly diseases ofaging), immunological disorders, infertility (or fertility), and others.

[0326] B) Treatment of Cancer

[0327] The present invention provides methods and compositions forreducing telomerase activity in tumor cells and for treating cancer.Compositions include antisense oligonucleotides, peptides, gene therapyvectors encoding antisense oligonucleotides or activity alteringproteins, and anti-hTRT antibodies. Cancer cells (e.g., malignant tumorcells) that express telomerase activity (telomerase-positive cells) canbe mortalized by decreasing or inhibiting the endogenous telomeraseactivity. Moreover, because telomerase levels correlate with diseasecharacteristics such as metastatic potential (e.g., U.S. Pat. Nos.5,639,613; 5,648,215; 5,489,508; Pandita et al., 1996, Proc. Am. Ass.Cancer Res. 37:559), any reduction in telomerase activity could reducethe aggressive nature of a cancer to a more manageable disease state(increasing the efficacy of traditional interventions).

[0328] The invention provides compositions and methods useful fortreatment of cancers of any of a wide variety of types, including solidtumors and leukemias. Types of cancer that may be treated include (butare not limited to): adenocarcinoma of the breast, prostate, and colon;all forms of bronchogenic carcinoma of the lung; myeloid; melanoma;hepatoma; neuroblastoma; papilloma; apudoma; choristoma; branchioma;malignant carcinoid syndrome; carcinoid heart disease; carcinoma (e.g.,Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor,in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell,papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, andtransitional cell), histiocytic disorders; leukemia (e.g., B-cell,mixed-cell, null-cell, T-cell, T-cell chronic, HTLV-II-associated,lyphocytic acute, lymphocytic chronic, mast-cell, and myeloid);histiocytosis malignant; Hodgkin's disease; immunoproliferative small;non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma;chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giantcell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;myxosarcoma; osteoma; osteosarcoma; Ewing's sarcoma; synovioma;adenofibroma; adenolymphoma; carcinosarcoma; chordoma;craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma;myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma;trophoblastic tumor; adenocarcinoma; adenoma; cholangioma;cholesteatoma; cylindroma; cystadenocarcinoma; cystadenoma; granulosacell tumor; gynandroblastoma; hepatoma; hidradenoma; islet cell tumor;leydig cell tumor; papilloma; sertoli cell tumor; theca cell tumor;leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma; rhabdomyoma;rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma;meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma;neuroma; paraganglioma; paraganglioma nonchromaffin; angiokeratoma;angiolymphoid hyperplasia with eosinophilia; angioma sclerosing;angiomatosis; glomangioma; hemangioendothelioma; hemangioma;hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma;lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma;cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma;leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma;ovarian carcinoma; rhabdomyosarcoma; sarcoma (e.g., Ewing's,experimental, Kaposi's, and mast-cell); neoplasms (e.g., bone, breast,digestive system, colorectal, liver, pancreatic, pituitary, testicular,orbital, head and neck, central nervous system, acoustic, pelvic,respiratory tract, and urogenital); neurofibromatosis, and cervicaldysplasia). The invention provides compositions and methods useful fortreatment of other conditions in which cells have become immortalized orhyperproliferative, e.g., by disregulation (e.g., abnormally highexpression) of hTRT, telomerase enzyme, or telomerase activity.

[0329] The present invention further provides compositions and methodsfor prevention of cancers, including anti-hTRT vaccines, gene therapyvectors that prevent telomerase activation, and gene therapy vectorsthat result in specific death of telomerase-positive cells. In a relatedaspect, the gene replacement therapy methods described below may be usedfor “treating” a genetic predilection for cancers.

[0330] C) Treatment of Other Conditions

[0331] The present invention also provides compositions and methodsuseful for treatment of diseases and disease conditions (in addition tocancers) characterized by under- or over-expression of telomerase orhTRT gene products. Examples include: diseases of cell proliferation,diseases resulting from cell senescence (particularly diseases ofaging), immunological disorders, infertility, diseases of immunedysfunction, and others.

[0332] Certain diseases of aging are characterized by cellsenescence-associated changes due to reduced telomere length (comparedto younger cells), resulting from the absence (or much lower levels) oftelomerase activity in the cell. Decreased telomere length and decreasedreplicative capacity contribute to diseases such as those describedbelow. Telomerase activity and telomere length can be increased by, forexample, increasing levels of hTRT gene products (protein and mRNA) inthe cell. A partial listing of conditions associated with cellularsenescence in which hTRT expression can be therapeutic includesAlzheimer's disease, Parkinson's disease, Huntington's disease, andstroke; age-related diseases of the integument such as dermal atrophy,elastolysis and skin wrinkling, sebaceous gland hyperplasia, senilelentigo, graying of hair and hair loss, chronic skin ulcers, andage-related impairment of wound healing; degenerative joint disease;osteoporosis; age-related immune system impairment (e.g., involvingcells such as B and T lymphocytes, monocytes, neutrophils, eosinophils,basophils, NK cells and their respective progenitors); age-relateddiseases of the vascular system including atherosclerosis,calcification, thrombosis, and aneurysms; diabetes, muscle atrophy,respiratory diseases, diseases of the liver and GI tract, metabolicdiseases, endocrine diseases (e.g., disorders of the pituitary andadrenal gland), reproductive diseases, and age-related maculardegeneration. These diseases and conditions can be treated by increasingthe levels of hTRT gene products in the cell to increase telomerelength, thereby restoring or imparting greater replicative capacity tothe cell. Such methods can be carried out on cells cultured ex vivo orcells in vivo. In one embodiment, the cells are first treated toactivate telomerase and lengthen telomeres, and then treated toinactivate the hTRT gene and telomerase activity. In a preferredembodiment, telomerase activity is generated by a vector of theinvention in an embryonic germ or stem cell prior to or duringdifferentiation.

[0333] The present invention also provides methods and compositionuseful for treating infertility. Human germline cells (e.g.,spermatogonia cells, their progenitors or descendants) are capable ofindefinite proliferation and characterized by high telomerase activity.Abnormal or diminished levels of hTRT gene products can result, forexample, in inadequate or abnormal production of spermatozoa, leading toinfertility or disorders of reproduction. Accordingly,“telomerase-based” infertility can be treated using the methods andcompositions described herein to increase telomerase levels. Similarly,because inhibition of telomerase may negatively impact spermatogenesis,oogenesis, and sperm and egg viability, the telomerase inhibitorycompositions of the invention can have contraceptive effects when usedto reduce hTRT gene product levels in germline cells.

[0334] Further, the invention provides methods and composition usefulfor decreasing the proliferative potential of telomerase-positive cellssuch as activated lymphocytes and hematopoietic stem cells by reducingtelomerase activity. Thus, the invention provide means for effectingimmunosuppression. Conversely, the methods and reagents of the inventionare useful for increasing telomerase activity and proliferativepotential in cells, such as stem cells, that express a low level oftelomerase or no telomerase prior to therapeutic intervention.

[0335] D) Modes of Intervention

[0336] As is clear from the foregoing discussion, modulation of thelevel of telomerase or telomerase activity of a cell can have a profoundeffect on the proliferative potential of the cell, and so has greatutility in treatment of disease. As is also clear, this modulation maybe either a decrease in telomerase activity or an increase in activity.The telomerase modulatory molecules of the invention can act through anumber of mechanisms; some of these are described in this and thefollowing subsections to aid the practitioner in selecting therapeuticagents. However, applicants do not intend to be limited to anyparticular mechanism of action for the novel therapeutic compounds,compositions and methods described herein.

[0337] Telomerase activity may be decreased through any of severalmechanisms or combinations of mechanisms. One mechanism is the reductionof hTRT gene expression to reduce telomerase activity. This reductioncan be at the level of transcription of the hTRT gene into mRNA,processing (e.g., splicing), nuclear transport or stability of mRNA,translation of mRNA to produce hTRT protein, or stability and functionof hTRT protein. Another mechanism is interference with one or moreactivities of telomerase (e.g., the reverse transcriptase catalyticactivity, or the hTR-binding activity) using inhibitory nucleic acids,polypeptides, or other agents (e.g., mimetics, small molecules, drugsand pro-drugs) that can be identified using the methods, or are providedby compositions, disclosed herein. Other mechanisms includesequestration of hTR and/or telomerase associated proteins, andinterference with the assembly of the telomerase RNP from its componentsubunits. In a related mechanism, an hTRT promoter sequence is operablylinked to a gene encoding a toxin and introduced into a cell; if or whenhTRT transcriptional activators are expressed or activated in the cell,the toxin will be expressed, resulting in specific cell killing.

[0338] A related method for reducing the proliferative capacity of acell involves introducing an hTRT variant with low fidelity (i.e., onewith a high, e.g., greater than 1%, error rate) such that aberranttelomeric repeats are synthesized. These aberrant repeats affecttelomere protein binding and lead to chromosomal rearrangements andaberrations and/or lead to cell death.

[0339] Similarly, telomerase activity may be increased through any ofseveral mechanisms, or a combination of mechanisms. These includeincreasing the amount of hTRT in a cell. Usually this is carried out byintroducing an hTRT polypeptide-encoding polynucleotide into the cell(e.g., a recombinantly produced polypeptide comprising an hTRT DNAsequence operably linked to a promoter, or a stable hTRT mRNA).Alternatively, a catalytically active hTRT polypeptide can itself beintroduced into a cell or tissue, e.g., by microinjection or other meansknown in the art. In other mechanisms, expression from the endogenoushTRT gene or the stability of hTRT gene products in the cell can beincreased. Telomerase activity in a cell can also be increased byinterfering with the interaction of endogenous telomerase inhibitors andthe telomerase RNP, or endogenous hTRT transcription repressors and thehTRT gene; by increasing expression or activity of hTRT transcriptionactivators; and other means apparent to those of skill upon review ofthis disclosure.

[0340] E) Intervention Agents

[0341] 1) TRT Proteins & Peptides

[0342] In one embodiment, the invention provides telomerase modulatorypolypeptides (i.e., proteins, polypeptides, and peptides) that increaseor reduce telomerase activity which can be introduced into a target celldirectly (e.g., by injection, liposome-mediated fusion, application of ahydrogel to the tumor [e.g., melanoma] surface, fusion or attachment toherpes virus structural protein VP22, and other means described hereinand known in the art). In a second embodiment, telomerase modulatoryproteins and peptides of the invention are expressed in a cell byintroducing a nucleic acid (e.g., a DNA expression vector or mRNA)encoding the desired protein or peptide into the cell. Expression may beeither constitutive or inducible depending on the vector and choice ofpromoter (see discussion below). Messenger RNA preparations encodinghTRT are especially useful when only transient expression (e.g.,transient activation of telomerase) is desired. Methods for introductionand expression of nucleic acids into a cell are well known in the art(also, see elsewhere in this specification, e.g., sections onoligonucleotides, gene therapy methods).

[0343] In one aspect of the invention, a telomerase modulatorypolypeptide that increases telomerase activity in a cell is provided. Inone embodiment, the polypeptide is a catalytically active hTRTpolypeptide capable of directing the synthesis (in conjunction with anRNA template such as hTR) of human telomeric DNA. This activity can bemeasured, as discussed above, e.g., using a telomerase activity assaysuch as a TRAP assay. In one embodiment, the polypeptide is afull-length hTRT protein, having a sequence of, or substantiallyidentical to, the sequence of 1132 residues of SEQ ID NO:2. In anotherembodiment, the polypeptide is a variant of the hTRT protein of SEQ IDNO:2, such as a fusion polypeptide, derivatized polypeptide, truncatedpolypeptide, conservatively substituted polypeptide, activity-modifiedpolypeptide, or the like. A fusion or derivatized protein may include atargeting moiety that increases the ability of the polypeptide totraverse a cell membrane or causes the polypeptide to be delivered to aspecified cell type (e.g., liver cells or tumor cells) preferentially orcell compartment (e.g., nuclear compartment) preferentially. Examples oftargeting moieties include lipid tails, amino acid sequences such asantennapoedia peptide or a nuclear localization signal (NLS; e.g.,Xenopus nucleoplasmin Robbins et al., 1991, Cell 64:615). Naturallyoccurring hTRT protein (e.g., having a sequence of, or substantiallyidentical to, SEQ ID NO:2) acts in the cell nucleus. Thus, it is likelythat one or more subsequences of SEQ ID NO:2, such as residues 193-196(PRRR SEQ ID NO:541) and residues 235-240 (PKRPRR SEQ ID NO: 542) act asa nuclear localization signal. The small regions are likely NLSs basedon the observation that many NLSs comprise a 4 residue pattern composedof basic amino acids (K or R), or composed of three basic amino acids (Kor R) and H or P; a pattern starting with P and followed within 3residues by a basic segment containing 3 K or R residues out of 4residues (see, e.g., Nakai et al., 1992, Genomics 14:897). Deletion ofone or both of these sequences and/or additional localization sequencesis expected to interfere with hTRT transport to the nucleus and/orincrease hTRT turnover, and is useful for preventing access oftelomerase to its nuclear substrates and decreasing proliferativepotential. Moreover, a variant hTRT polypeptide lacking NLS may assembleinto an RNP that will not be able to maintain telomere length, becausethe resulting enzyme cannot enter the nucleus.

[0344] The hTRT polypeptides of the invention will typically beassociated in the target cell with a telomerase RNA, such as hTR,especially when they are used to increase telomerase activity in a cell.In one embodiment, an introduced hTRT polypeptide associates with anendogenous hTR to form a catalytically active RNP (e.g., an RNPcomprising the hTR and a full-length polypeptide having a sequence ofSEQ ID NO:2). The RNP so-formed may also associate with other, e.g.,telomerase-associated, proteins. In other embodiments, telomerase RNP(containing hTRT protein, hTR and optionally other components) isintroduced as a complex to the target cell.

[0345] In a related embodiment, an hTRT expression vector is introducedinto a cell (or progeny of a cell) into which a telomerase RNA (e.g.,hTR) expression vector is simultaneously, subsequently or has beenpreviously introduced. In this embodiment, hTRT protein and telomeraseRNA are coexpressed in the cell and assemble to form a telomerase RNP. Apreferred telomerase RNA is hTR. An expression vector useful forexpression of hTR in a cell is described supra (see U.S. Pat. No.5,583,016). In yet another embodiment, the hTRT polypeptide and hTR RNA(or equivalent) are associated in vitro to form a complex, which is thenintroduced into the target cells, e.g., by liposome mediated transfer.

[0346] In another aspect, the invention provides hTRT polypeptidesuseful for reducing telomerase activity in a cell. As above, these“inhibitory” polypeptides can be introduced directly, or by expressionof recombinant nucleic acids in the cell. It will be recognized thatpeptide mimetics or polypeptides comprising nonstandard amino acids(i.e., other than the 20 amino acids encoded by the genetic code ortheir normal derivatives) will typically be introduced directly.

[0347] In one embodiment, inhibition of telomerase activity results fromthe sequestration of a component required for accurate telomereelongation. Examples of such components are hTRT and hTR. Thus,administration of a polypeptide that binds hTR, but which does not havetelomerase catalytic activity, can reduce endogenous telomerase activityin the cell. In a related embodiment, the hTRT polypeptide may bind acell component other than hTR, such as one or more telomerase-associatedproteins, thereby interfering with telomerase activity in the cell.

[0348] In another embodiment, hTRT polypeptides of the inventioninterfere (e.g., by competition) with the interaction of endogenouslyexpressed hTRT protein and another cellular component required fortelomerase function, such as hTR, telomeric DNA, telomerase-associatedproteins, telomere-associated proteins, telomeres, cell cycle controlproteins, DNA repair enzymes, histone or non-histone chromosomalproteins, or others.

[0349] In selecting molecules (e.g., polypeptides) of the invention thataffect the interaction of endogenously expressed hTRT protein and othercellular components, one may prefer molecules that include one or moreof the conserved motifs of the hTRT protein, as described herein. Theevolutionary conservation of these regions indicates the importantfunction in the proper functioning of human telomerase contributed bythese motifs, and the motifs are thus generally useful sites forchanging hTRT protein function to create variant hTRT proteins of theinvention. Thus, variant hTRT polypeptides having mutations in conservedmotifs will be particularly useful for some applications of theinvention.

[0350] In another embodiment, expression of the endogenous hTRT gene isrepressed by introduction into the cell of a large amount of hTRTpolypeptide (e.g., typically at least about 2-fold more than theendogenous level, more often at least about 10- to about 100-fold) whichacts via a feedback loop to inhibit transcription of the hTRT gene,processing of the hTRT pre-mRNA, translation of the hTRT mRNA, orassembly and transport of the telomerase RNP.

[0351] 2) Oligonucleotides

[0352] a) Antisense Constructs

[0353] The invention provides methods and antisense oligonucleotide orpolynucleotide reagents which can be used to reduce expression of hTRTgene products in vitro or in vivo. Administration of the antisensereagents of the invention to a target cell results in reduced telomeraseactivity, and is particularly useful for treatment of diseasescharacterized by high telomerase activity (e.g., cancers). Withoutintending to be limited to any particular mechanism, it is believed thatantisense oligonucleotides bind to, and interfere with the translationof, the sense hTRT mRNA. Alternatively, the antisense molecule mayrender the hTRT mRNA susceptible to nuclease digestion, interfere withtranscription, interfere with processing, localization or otherwise withRNA precursors (“pre-mRNA”), repress transcription of mRNA from the hTRTgene, or act through some other mechanism. However, the particularmechanism by which the antisense molecule reduces hTRT expression is notcritical.

[0354] The antisense polynucleotides of the invention comprise anantisense sequence of at least 7 to 10 to typically 20 or morenucleotides that specifically hybridize to a sequence from mRNA encodinghTRT or mRNA transcribed from the hTRT gene. More often, the antisensepolynucleotide of the invention is from about 10 to about 50 nucleotidesin length or from about 14 to about 35 nucleotides in length. In otherembodiments, antisense polynucleotides are polynucleotides of less thanabout 100 nucleotides or less than about 200 nucleotides. In general,the antisense polynucleotide should be long enough to form a stableduplex but short enough, depending on the mode of delivery, toadminister in vivo, if desired. The minimum length of a polynucleotiderequired for specific hybridization to a target sequence depends onseveral factors, such as G/C content, positioning of mismatched bases(if any), degree of uniqueness of the sequence as compared to thepopulation of target polynucleotides, and chemical nature of thepolynucleotide (e.g., methylphosphonate backbone, peptide nucleic acid,phosphorothioate), among other factors.

[0355] Generally, to assure specific hybridization, the antisensesequence is substantially complementary to the target hTRT mRNAsequence. In certain embodiments, the antisense sequence is exactlycomplementary to the target sequence. The antisense polynucleotides mayalso include, however, nucleotide substitutions, additions, deletions,transitions, transpositions, or modifications, or other nucleic acidsequences or non-nucleic acid moieties so long as specific binding tothe relevant target sequence corresponding to hTRT RNA or its gene isretained as a functional property of the polynucleotide.

[0356] In one embodiment, the antisense sequence is complementary torelatively accessible sequences of the hTRT mRNA (e.g., relativelydevoid of secondary structure). This can be determined by analyzingpredicted RNA secondary structures using, for example, the MFOLD program(Genetics Computer Group, Madison Wis.) and testing in vitro or in vivoas is known in the art. Examples of oligonucleotides that may be testedin cells for antisense suppression of hTRT function are those capable ofhybridizing to (i.e., substantially complementary to) the followingpositions from SEQ ID NO:1: 40-60; 260-280; 500-520; 770-790; 885-905;1000-1020; 1300-1320; 1520-1540; 2110-2130; 2295-2315; 2450-2470;2670-2690; 3080-3110; 3140-3160; and 3690-3710. Another useful methodfor identifying effective antisense compositions uses combinatorialarrays of oligonucleotides (see, e.g., Milner et al., 1997, NatureBiotechnology 15:537).

[0357] The invention also provides an antisense polynucleotide that hassequences in addition to the antisense sequence (i.e., in addition toanti-hTRT-sense sequence). In this case, the antisense sequence iscontained within a polynucleotide of longer sequence. In anotherembodiment, the sequence of the polynucleotide consists essentially of,or is, the antisense sequence.

[0358] The antisense nucleic acids (DNA, RNA, modified, analogues, andthe like) can be made using any suitable method for producing a nucleicacid, such as the chemical synthesis and recombinant methods disclosedherein. In one embodiment, for example, antisense RNA molecules of theinvention may be prepared by de novo chemical synthesis or by cloning.For example, an antisense RNA that hybridizes to hTRT mRNA can be madeby inserting (ligating) an hTRT DNA sequence (e.g., SEQ ID NO:1, orfragment thereof) in reverse orientation operably linked to a promoterin a vector (e.g., plasmid). Provided that the promoter and, preferablytermination and polyadenylation signals, are properly positioned, thestrand of the inserted sequence corresponding to the noncoding strandwill be transcribed and act as an antisense oligonucleotide of theinvention.

[0359] The antisense oligonucleotides of the invention can be used toinhibit telomerase activity in cell-free extracts, cells, and animals,including mammals and humans. For example, the phosphorothioateantisense oligonucleotides: A) 5′-GGCATCGCGGGGGTGGCCGGG SEQ ID NO:506 B)5′-CAGCGGGGAGCGCGCGGCATC SEQ ID NO:521 C) 5′-CAGCACCTCGCGGTAGTGGCT SEQID NO:522 D) 5′-GGACACCTGGCGGAAGGAGGG SEQ ID NO:507

[0360] can be used to inhibit telomerase activity. At 10 micromolarconcentration each oligonucleotide, mixtures of oligonucleotides A andB; A, B, C, and D; and A, C, and D inhibited telomerase activity in 293cells when treated once per day for seven days. Inhibition was alsoobserved when an antisense hTR molecule (5′-GCTCTAGAATGAAGGGTG-3′; 3′SEQ ID NO:543) was used in combination with oligonucleotides A, B, andC; A, B, and D; and A and C. Useful control oligonucleotides in suchexperiments include: S1) 5′-GCGACGACTGACATTGGCCGG SEQ ID NO:544 S2)5′-GGCTCGAAGTAGCACCGGTGC SEQ ID NO:545 S3) 5′-GTGGGAACAGGCCGATGTCCC SEQID NO:546

[0361] To determine the optimum antisense oligonucleotide of theinvention for the particular application of interest, one can perform ascan using antisense oligonucleotide sets of the invention. Oneillustrative set is the set of 30-mer oligonucleotides that span thehTRT mRNA and are offset one from the next by fifteen nucleotides (i.e.,ON1 corresponds to positions 1-30 and is TCCCACGTGCGCAGCAGGACGCAGCGCTGC(SEQ ID NO:547), ON2 corresponds to positions 16-45 and isGCCGGGGCCAGGGCTTCCCACGTGCGCAGC (SEQ ID NO:548), and ON3 corresponds topositions 31-60 and is GGCATCGCGGGGGTGGCCGGGGCCAGGGCT (SEQ ID NO:549),and so on to the end of the mRNA). Each member of this set can be testedfor inhibitory activity as disclosed herein. Those oligonucleotides thatshow inhibitory activity under the conditions of interest then identifya region of interest, and other oligonucleotides of the inventioncorresponding to the region of interest (i.e., 8-mers, 10-mers, 15-mers,and so on) can be tested to identify the oligonucleotide with thepreferred activity for the application.

[0362] Exemplary antisense oligonucleotides include 5′-GGCATCGCGGGGGTGGCCGGGGCCAGGGCT-3′ (SEQ ID NO:722) (corresponding to nucleotidepositions 31-60 of hTRT); 5′-GCGCA GCGTGCCAGCAGGTGAACCAGCACG-3′ (SEQ IDNO:723) (corresponding to positions 496-525);5′-GCCCGTTCGCATCCCAGACGCCTTCGGGGT-3′ (SEQ ID NO:724) (corresponding topositions 631-660); and 5′-ACGCTATGGTTCCAGGCCCGTTCGCATCCC-3′ (SEQ IDNO:725) (corresponding to positions 646-675). When ACHN cells (NCI#503755) or 293 cells were treated for three days with 10 μM ofphosphorothioate oligonucleotides with any of the four sequences supra,inhibition of telomerase activity by about 50%-90% (compared to controluntreated cells) as measured by a TRAP assay, was observed.

[0363] For general methods relating to antisense polynucleotides, seeANTISENSE RNA AND DNA, (1988), D. A. Melton, Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). See also, Dagle et al., 1991,Nucleic Acids Research, 19:1805. For a review of antisense therapy, see,e.g., Uhlmann et al., Chem. Reviews, 90:543-584 (1990).

[0364] b) Triplex Oligo- and Polynucleotides

[0365] The present invention provides oligo- and polynucleotides (e.g.,DNA, RNA, PNA or the like) that bind to double-stranded or duplex hTRTnucleic acids (e.g., in a folded region of the hTRT RNA or in the hTRTgene), forming a triple helix-containing, or “triplex” nucleic acid.Triple helix formation results in inhibition of hTRT expression by, forexample, preventing transcription of the hTRT gene, thus reducing oreliminating telomerase activity in a cell. Without intending to be boundby any particular mechanism, it is believed that triple helix pairingcompromises the ability of the double helix to open sufficiently for thebinding of polymerases, transcription factors, or regulatory moleculesto occur.

[0366] Triplex oligo- and polynucleotides of the invention areconstructed using the base-pairing rules of triple helix formation (see,e.g., Cheng et al., 1988, J. Biol. Chem. 263: 15110; Ferrin andCamerini-Otero, 1991, Science 354:1494; Ramdas et al., 1989, J. Biol.Chem. 264:17395; Strobel et al., 1991, Science 254:1639; and Rigas etal., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 9591; each of which isincorporated herein by reference) and the hTRT mRNA and/or genesequence. Typically, the triplex-forming oligonucleotides of theinvention comprise a specific sequence of from about 10 to at leastabout 25 nucleotides or longer “complementary” to a specific sequence inthe hTRT RNA or gene (i.e., large enough to form a stable triple helix,but small enough, depending on the mode of delivery, to administer invivo, if desired). In this context, “complementary” means able to form astable triple helix. In one embodiment, oligonucleotides are designed tobind specifically to the regulatory regions of the hTRT gene (e.g., thehTRT 5′-flanking sequence, promoters, and enhancers) or to thetranscription initiation site, (e.g., between −10 and +10 from thetranscription initiation site). For a review of recent therapeuticadvances using triplex DNA, see Gee et al., in Huber and Carr, 1994,MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co, Mt KiscoN.Y. and Rininsland et al., 1997, Proc. Natl. Acad. Sci. USA 94:5854,which are both incorporated herein by reference.

[0367] c) Ribozymes

[0368] The present invention also provides ribozymes useful forinhibition of telomerase activity. The ribozymes of the invention bindand specifically cleave and inactivate hTRT mRNA. Useful ribozymes cancomprise 5′- and 3′-terminal sequences complementary to the hTRT mRNAand can be engineered by one of skill on the basis of the hTRT mRNAsequence disclosed herein (see PCT publication WO 93/23572, supra).Ribozymes of the invention include those having characteristics of groupI intron ribozymes (Cech, 1995, Biotechnology 13:323) and others ofhammerhead ribozymes (Edgington, 1992, Biotechnology 10:256).

[0369] Ribozymes of the invention include those having cleavage sitessuch as GUA, GUU and GUC. Other optimum cleavage sites forribozyme-mediated inhibition of telomerase activity in accordance withthe present invention include those described in PCT publications WO94/02595 and WO 93/23569, both incorporated herein by reference. ShortRNA oligonucleotides between 15 and 20 ribonucleotides in lengthcorresponding to the region of the target hTRT gene containing thecleavage site can be evaluated for secondary structural features thatmay render the oligonucleotide more desirable. The suitability ofcleavage sites may also be evaluated by testing accessibility tohybridization with complementary oligonucleotides using ribonucleaseprotection assays, or by testing for in vitro ribozyme activity inaccordance with standard procedures known in the art.

[0370] As described by Hu et al., PCT publication WO 94/03596,incorporated herein by reference, antisense and ribozyme functions canbe combined in a single oligonucleotide. Moreover, ribozymes cancomprise one or more modified nucleotides or modified linkages betweennucleotides, as described above in conjunction with the description ofillustrative antisense oligonucleotides of the invention.

[0371] In one embodiment, the ribozymes of the invention are generatedin vitro and introduced into a cell or patient. In another embodiment,gene therapy methods are used for expression of ribozymes in a targetcell ex vivo or in vivo.

[0372] d) Administration of Oligonucleotides

[0373] Typically, the therapeutic methods of the invention involve theadministration of an oligonucleotide that functions to inhibit orstimulate telomerase activity under in vivo physiological conditions,and is relatively stable under those conditions for a period of timesufficient for a therapeutic effect. As noted above, modified nucleicacids may be useful in imparting such stability, as well as fortargeting delivery of the oligonucleotide to the desired tissue, organ,or cell.

[0374] Oligo- and poly-nucleotides can be delivered directly as a drugin a suitable pharmaceutical formulation, or indirectly by means ofintroducing a nucleic acid into a cell, including liposomes,immunoliposomes, ballistics, direct uptake into cells, and the like asdescribed herein. For treatment of disease, the oligonucleotides of theinvention will be administered to a patient in a therapeuticallyeffective amount. A therapeutically effective amount is an amountsufficient to ameliorate the symptoms of the disease or modulatetelomerase activity in the target cell, e.g., as can be measured using aTRAP assay or other suitable assay of telomerase biological function.Methods useful for delivery of oligonucleotides for therapeutic purposesare described in U.S. Pat. No. 5,272,065, incorporated herein byreference. Other details of administration of pharmaceutically activecompounds are provided below. In another embodiment, oligo- andpoly-nucleotides can be delivered using gene therapy and recombinant DNAexpression plasmids of the invention.

[0375] 3) Gene Therapy

[0376] Gene therapy refers to the introduction of an otherwise exogenouspolynucleotide which produces a medically useful phenotypic effect uponthe (typically) mammalian cell(s) into which it is transferred. In oneaspect, the present invention provides gene therapy methods andcompositions for treatment of telomerase-associated conditions. Inillustrative embodiments, gene therapy involves introducing into a cella vector that expresses an hTRT gene product (such as an hTRT proteinsubstantially similar to the hTRT polypeptide having a sequence ofSEQUENCE ID NO: 2, e.g., to increase telomerase activity, or aninhibitory hTRT polypeptide to reduce activity), expresses a nucleicacid having an hTRT gene or mRNA sequence (such as an antisense RNA,e.g., to reduce telomerase activity), expresses a polypeptide orpolynucleotide that otherwise affects expression of hTRT gene products(e.g., a ribozyme directed to hTRT mRNA to reduce telomerase activity),or replaces or disrupts an endogenous hTRT sequence (e.g., genereplacement and “gene knockout,” respectively). Numerous otherembodiments will be evident to one of skill upon review of thedisclosure herein. In one embodiment, a vector encoding hTR is alsointroduced. In another embodiment, vectors encodingtelomerase-associated proteins are also introduced with or without avector for hTR.

[0377] Vectors useful in hTRT gene therapy can be viral or nonviral, andinclude those described supra in relation to the hTRT expression systemsof the invention. It will be understood by those of skill in the artthat gene therapy vectors may comprise promoters and other regulatory orprocessing sequences, such as are described in this disclosure. Usuallythe vector will comprise a promoter and, optionally, an enhancer(separate from any contained within the promoter sequences) that serveto drive transcription of an oligoribonucleotide, as well as otherregulatory elements that provide for episomal maintenance or chromosomalintegration and for high-level transcription, if desired. A plasmiduseful for gene therapy can comprise other functional elements, such asselectable markers, identification regions, and other sequences. Theadditional sequences can have roles in conferring stability both outsideand within a cell, targeting delivery of hTRT nucleotide sequences(sense or antisense) to a specified organ, tissue, or cell population,mediating entry into a cell, mediating entry into the nucleus of a celland/or mediating integration within nuclear DNA. For example,aptamer-like DNA structures, or other protein binding moieties sites canbe used to mediate binding of a vector to cell surface receptors or toserum proteins that bind to a receptor thereby increasing the efficiencyof DNA transfer into the cell. Other DNA sites and structures candirectly or indirectly bind to receptors in the nuclear membrane or toother proteins that go into the nucleus, thereby facilitating nuclearuptake of a vector. Other DNA sequences can directly or indirectlyaffect the efficiency of integration.

[0378] Suitable gene therapy vectors may, or may not, have an origin ofreplication. For example, it is useful to include an origin ofreplication in a vector for propagation of the vector prior toadministration to a patient. However, the origin of replication canoften be removed before administration if the vector is designed tointegrate into host chromosomal DNA or bind to host mRNA or DNA. In somesituations (e.g., tumor cells) it may not be necessary for the exogenousDNA to integrate stably into the transduced cell, because transientexpression may suffice to kill the tumor cells.

[0379] As noted, the present invention also provides methods andreagents for gene replacement therapy (i.e., replacement by homologousrecombination of an endogenous hTRT gene with a recombinant gene).Vectors specifically designed for integration by homologousrecombination may be used. Important factors for optimizing homologousrecombination include the degree of sequence identity and length ofhomology to chromosomal sequences. The specific sequence mediatinghomologous recombination is also important, because integration occursmuch more easily in transcriptionally active DNA. Methods and materialsfor constructing homologous targeting constructs are described by e.g.,Mansour et al., 1988, Nature 336: 348; Bradley et al., 1992,Bio/Technology 10: 534. See also, U.S. Pat. Nos. 5,627,059; 5,487,992;5,631,153; and 5,464,764. In one embodiment, gene replacement therapyinvolves altering or replacing all or a portion of the regulatorysequences controlling expression of the hTRT gene that is to beregulated. For example, the hTRT promoter sequences (e.g., such as arefound in SEQ ID NO:6) may be disrupted (to decrease hTRT expression orto abolish a transcriptional control site) or an exogenous promoter(e.g., to increase hTRT expression) substituted.

[0380] The invention also provides methods and reagents for hTRT “geneknockout” (i.e., deletion or disruption by homologous recombination ofan endogenous hTRT gene using a recombinantly produced vector). In geneknockout, the targeted sequences can be regulatory sequences (e.g., thehTRT promoter), or RNA or protein coding sequences. The use ofhomologous recombination to alter expression of endogenous genes isdescribed in detail in U.S. Pat. No. 5,272,071 (and the U.S. patentscited supra), WO 91/09955, WO 93/09222, WO 96/29411, WO 95/31560, and WO91/12650. See also, Moynahan et al., 1996, Hum. Mol. Genet. 5:875.

[0381] The invention further provides methods for specifically killingtelomerase-positive cells, or preventing transformation of telomerasenegative cells to a telomerase positive state, using the hTRT genepromoter to regulate expression of a protein toxic to the cell. As shownin Example 14, an hTRT promoter sequence may be operably linked to areporter gene such that activation of the promoter results in expressionof the protein encoded by the reporter gene. If, instead of a reporterprotein, the encoded protein is toxic to the cell, activation of thepromoter leads to cell morbidity or death. In one embodiment of thepresent invention, a vector comprising an hTRT promoter operably linkedto a gene encoding a toxic protein is introduced into cells, such ashuman cells, e.g., cells in a human patient, resulting in cell death ofcells in which hTRT promoter activating factors are expressed, such ascancer cells. In a related embodiment, the encoded protein is not itselftoxic to a cell, but encodes an activity that renders the cell sensitiveto an otherwise nontoxic drug. For example, tumors can be treated byintroducing an hTRT-promoter-Herpes thymidine kinase (TK) gene fusionconstruct into tumor cells, and administering gancyclovir or theequivalent (see, e.g., Moolton and Wells, 1990, J. Nat'l Canc. Inst.82:297). The art knows of numerous other suitable toxic or potentiallytoxic proteins and systems (using promoter sequences other that hTRT)that may be modified and applied in accordance with the presentinvention by one of skill in the art upon review of this disclosure.

[0382] Gene therapy vectors may be introduced into cells or tissues invivo, in vitro or ex vivo. For ex vivo therapy, vectors may beintroduced into cells, e.g., stem cells, taken from the patient andclonally propagated for autologous transplant back into the same patient(see, e.g., U.S. Pat. Nos. 5,399,493 and 5,437,994, the disclosures ofwhich are herein incorporated by reference). Cells that can be targetedfor hTRT gene therapy aimed at increasing the telomerase activity of atarget cell include, but are not limited to, embryonic stem or germcells, particularly primate or human cells, as noted supra,hematopoietic stem cells (AIDS and post-chemotherapy), vascularendothelial cells (cardiac and cerebral vascular disease), skinfibroblasts and basal skin keratinocytes (wound healing and burns),chondrocytes (arthritis), brain astrocytes and microglial cells(Alzheimer's Disease), osteoblasts (osteoporosis), retinal cells (eyediseases), and pancreatic islet cells (Type I diabetes) and any of thecells listed in Table 3, infra, as well as any other cell types known todivide.

[0383] In one embodiment of the invention, an inducible promoteroperably linked to a TRT, such as hTRT, coding sequence (or variant) isused to modulate the proliferative capacity of cells in vivo or invitro. In a particular embodiment, for example, insulin-producingpancreatic cells transfected with an hTRT expression vector under thecontrol of an inducible promoter are introduced into a patient. Theproliferative capacity of the cells can then be controlled byadministration to the patient of the promoter activating agent (e.g.,tetracycline) to enable the cells to multiply more than otherwise wouldhave been possible. Cell proliferation can then be terminated,continued, or reinitiated as desired by the treating physician.

[0384] 4) Vaccines and Antibodies

[0385] Immuogenic peptides or polypeptides having an hTRT sequence canbe used to elicit an anti-hTRT immune response in a patient (i.e., actas a vaccine). Exemplary immunogenic hTRT peptides and polypeptides aredescribed infra in Examples 6 and 8. An immune response can also beraised by delivery of plasmid vectors encoding the polypeptide ofinterest (i.e., administration of “naked DNA”). The nucleic acids ofinterest can be delivered by injection, liposomes, or other means ofadministration. In one embodiment, immunization modes that elicit in thesubject a Class I MHC restricted cytotoxic lymphocyte response againsttelomerase expressing cells are chosen. Once immunized, the individualor animal will elicit a heightened immune response against cellsexpressing high levels of telomerase (e.g., malignant cells).

[0386] Anti-hTRT antibodies, e.g., murine, human, or humanizedmonoclonal antibodies may also be administered to a patient (e.g.,passive immunization) to effect an immune response againsttelomerase-expressing cells.

[0387] F) Pharmaceutical Compositions

[0388] In related aspects, the invention provides pharmaceuticalcompositions that comprise hTRT oligo- and poly-nucleotides,polypeptides, and antibodies, agonists, antagonists, or inhibitors,alone or in combination with at least one other agent, such as astabilizing compound, diluent, carrier, or another active ingredient oragent.

[0389] The therapeutic agents of the invention may be administered inany sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Any of thesemolecules can be administered to a patient alone, or in combination withother agents, drugs or hormones, in pharmaceutical compositions where itis mixed with suitable excipient(s), adjuvants, and/or pharmaceuticallyacceptable carriers. In one embodiment of the present invention, thepharmaceutically acceptable carrier is pharmaceutically inert.

[0390] Administration of pharmaceutical compositions is accomplishedorally or parenterally. Methods of parenteral delivery include topical,intra-arterial (e.g., directly to the tumor), intramuscular,subcutaneous, intramedullary, intrathecal, intraventricular,intravenous, intraperitoneal, or intranasal administration. In additionto the active ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andother compounds that facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of “REMINGTON'S PHARMACEUTICAL SCIENCES” (Maack Publishing Co,Easton Pa.).

[0391] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc.,suitable for ingestion by the patient. See PCT publication WO 93/23572.

[0392] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable additional compounds, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers include, but are not limited to sugars, including lactose,sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato,or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; as well as proteins such asgelatin and collagen. If desired, disintegrating or solubilizing agentsmay be added, such as the cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate.

[0393] Dragee cores are provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage).

[0394] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

[0395] Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

[0396] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0397] The pharmaceutical compositions of the present invention may bemanufactured in a manner similar to that known in the art (e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses).

[0398] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0399] After pharmaceutical compositions comprising a compound of theinvention formulated in a acceptable carrier have been prepared, theycan be placed in an appropriate container and labeled for treatment ofan indicated condition. For administration of human telomerase proteinsand nucleic acids, such labeling would include amount, frequency andmethod of administration.

[0400] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose.“Therapeutically effective amount” or “pharmacologically effectiveamount” are well recognized phrases and refer to that amount of an agenteffective to produce the intended pharmacological result. Thus, atherapeutically effective amount is an amount sufficient to amelioratethe symptoms of the disease being treated. One useful assay inascertaining an effective amount for a given application (e.g., atherapeutically effective amount) is measuring the effect on telomeraseactivity in a target cell. The amount actually administered will bedependent upon the individual to which treatment is to be applied, andwill preferably be an optimized amount such that the desired effect isachieved without significant side-effects. The determination of atherapeutically effective dose is well within the capability of thoseskilled in the art.

[0401] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays or in any appropriateanimal model. The animal model is also used to achieve a desirableconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans.

[0402] A therapeutically effective amount refers to that amount ofprotein, polypeptide, peptide, antibody, oligo- or polynucleotide,agonist or antagonists which ameliorates the symptoms or condition.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals (e.g., ED₅₀, the dose therapeutically effective in 50% of thepopulation; and LD₅₀, the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, ED₅₀/LD₅₀. Pharmaceuticalcompositions which exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

[0403] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors which may be taken into accountinclude the severity of the disease state (e.g., tumor size andlocation; age, weight and gender of the patient; diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy). Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation. Guidance as to particular dosagesand methods of delivery is provided in the literature (see, U.S. Pat.Nos. 4,657,760; 5,206,344; and 5,225,212, herein incorporated byreference). Those skilled in the art will typically employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides can be specificto particular cells, conditions, locations, and the like.

[0404] VIII. Increasing Proliferative Capacity and Production ofImmortalized Cells, Cell Lines, and Animals

[0405] As discussed above, most vertebrate cells senesce after a finitenumber of divisions in culture (e.g., 50 to 100 divisions). Certainvariant cells, however, are able to divide indefinitely in culture(e.g., HeLa cells, 293 cells) and, for this reason, are useful forresearch and industrial applications. Usually these immortal cell linesare derived from spontaneously arising tumors, or by transformation byexposure to radiation or a tumor-inducing virus or chemical.Unfortunately, a limited selection of cell lines, especially human celllines representing differentiated cell function, is available. Moreover,the immortal cell lines presently available are characterized bychromosomal abnormalities (e.g., aneuploidy, gene rearrangements, ormutations). Further, many long-established cell lines are relativelyundifferentiated (e.g., they do not produce highly specialized productsof the sort that uniquely characterize particular tissues or organs).Thus, there is a need for new methods of generating immortal cells,especially human cells. As used herein, the term “immortalized cells” isnot limited to cells that proliferate indefinitely, but may also includecells with increased proliferative capacity compared to similarwild-type cells. Depending on the cell type, increased proliferativecapacity may mean proliferation for at least about 100, about 150, about200, or about 400 or more generations, or for at least about 6, about12, about 18, about 24 or about 36 or more months in in vitro culture.One use for immortalized cells is in production of natural proteins andrecombinant proteins (e.g., therapeutic polypeptides such aserythropoietin, human growth hormone, insulin, and the like), orantibodies, for which a stable, genetically normal cell line ispreferred. For production of some recombinant proteins, specialized celltypes may also be preferred (e.g., pancreatic cells for the productionof human insulin). Another use for immortalized cells or even mortalcells with increased proliferative capacity (relative to unmodifiedcells) is for introduction into a patient for gene therapy, or forreplacement of diseased or damaged cells or tissue. For example,autologous immune cells containing or expressing a, e.g., recombinanthTRT gene or polypeptide of the invention can be used for cellreplacement in a patient after aggressive cancer therapy, e.g., wholebody irradiation. Another use for immortalized cells is for ex vivoproduction of “artificial” tissues or organs (e.g., skin) fortherapeutic use. Another use for such cells is for screening orvalidation of drugs, such as telomerase-inhibiting drugs, or for use inproduction of vaccines or biological reagents. Additional uses of thecells of the invention will be apparent to those of skill.

[0406] The immortalized cells and cell lines, as well as those of merelyincreased replicative capacity, of the invention are made by increasingtelomerase activity in the cell. Any method disclosed herein forincreasing telomerase activity can be used. Thus, in one embodiment,cells are immortalized by increasing the amount of an hTRT polypeptidein the cell. In one embodiment, hTRT levels are increased by introducingan hTRT expression vector into the cell (with stable transfectionsometimes preferred). As discussed above, the hTRT coding sequence isusually operably linked to a promoter, which may be inducible orconstitutively active in the cell.

[0407] In one embodiment, a polynucleotide comprising a sequenceencoding a polypeptide of SEQ ID NO:2, which sequence is operably linkedto a promoter (e.g., a constitutively expressed promoter, e.g., asequence of SEQ ID NO:6, is introduced into the cell. In one embodimentthe polynucleotide comprises a sequence of SEQ ID NO:1. Preferably thepolynucleotide includes polyadenylation and termination signals. Inother embodiments, additional elements such as enhancers or othersdiscussed supra are included. In an alternative embodiment, thepolynucleotide does not include a promoter sequence, such sequence beingprovided by the target cell endogenous genome following integration(e.g., recombination, e.g., homologous recombination) of the introducedpolynucleotide. The polynucleotide may be introduced into the targetcell by any method, including any method disclosed herein, such aslipofection, electroporation, virosomes, liposomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA).

[0408] Using the methods of the invention, any vertebrate cell can becaused to have an increased proliferative capacity or even beimmortalized and sustained indefinitely in culture. In one embodimentthe cells are mammalian, with human cells preferred for manyapplications. Examples of human cells that can be immortalized includethose listed in Table 3.

[0409] It will be recognized that the “diagnostic” assays of theinvention described infra may be used to identify and characterize theimmortalized cells of the invention. TABLE 3 HUMAN CELLS IN WHICH hTRTEXPRESSION MAY BE INCREASED Keratinizing Epithelial Cells keratinocyteof epidermis (differentiating epidermal cell) basal cell of epidermis(stem cell) keratinocyte of fingernails and toenails basal cell of nailbed (stem cell) hair shaft cells medullary, cortical, cuticular;hair-root sheath cells, cuticular, of Huxley's layer, of Henle's layerexternal; hair matrix cell (stem cell) Cells of Wet Stratified BarrierEpithelia surface epithelial cell of stratified squamous epithelium oftongue, oral cavity, esophagus, anal canal, distal urethra, vagina basalcell of these epithelia (stem cell) cell of external corneal epitheliumcell of urinary epithelium (lining bladder and urinary ducts) EpithelialCells Specialized for Exocrine Secretion cells of salivary gland mucouscell (secretion rich in polysaccharide) serous cell (secretion rich inglycoprotein enzymes) cell of von Ebner's gland in tongue (secretion towash over taste buds) cell of mammary gland, secreting milk cell oflacrimal gland, secreting tears cell of ceruminous gland of ear,secreting wax cell of eccrine sweat gland, secreting glycoproteins (darkcell) cell of eccrine sweat gland, secreting small molecules (clearcell) cell of apocrine sweat gland (odoriferous secretion, sex- hormonesensitive) cell of gland of Moll in eyelid (specialized sweat gland)cell of sebaceous gland, secreting lipid-rich sebum cell of Bowman'sgland in nose (secretion to wash over olfactory epithelium) cell ofBrunner's gland in duodenum, secreting alkaline solution of mucus andenzymes cell of seminal vesicle, secreting components of seminal fluid,including fructose (as fuel for swimming sperm) cell of prostate gland,secreting other components of seminal fluid cell of bulbourethral gland,secreting mucus cell of Bartholin's gland, secreting vaginal lubricantcell of gland of Littre, secreting mucus cell of endometrium of uterus,secreting mainly carbohydrates isolated goblet cell of respiratory anddigestive tracts, secreting mucus mucous cell of lining of stomachzymogenic cell of gastric gland, secreting pepsinogen oxyntic cell ofgastric gland, secreting HCl acinar cell of pancreas, secretingdigestive enzymes and bicarbonate Paneth cell of small intestine,secreting lysozyme type II pneumocyte of lung, secreting surfactantClara cell of lung Cells specialized for Secretion of Hormones cells ofanterior pituitary, secreting growth hormone, follicle-stimulatinghormone, luteinizing hormone, prolactin, adrenocorticotropic hormone,and thyroid-stimulating hormone, cell of intermediate pituitary,secreting melanocyte-stimulating hormone cells of posterior pituitary,secreting oxytocin, vasopressin cells of gut, secreting serotonin,endorphin, somatostatin, gastrin, secretin, cholecystokinin, insulin andglucagon cells of thyroid gland, secreting thyroid hormone, calcitonincells of parathyroid gland, secreting parathyroid hormone, oxyphil cellcells of adrenal gland, secreting epinephrine, norepinephrine, andsteroid hormones; mineralocorticoids glucocorticoids cells of gonads,secreting testosterone (Leydig cell of testis) estrogen (theca internacell of ovarian follicle) progesterone (corpus luteum cell of rupturedovarian follicle) cells of juxtaglomerular apparatus of kidneyjuxtaglomerular cell (secreting renin) macula densa cell peripolar cellmesangial cell Epithelial Absorptive Cells in Gut, Exocrine Glands, andUrogenital Tract brush border cell of intestine (with microvilli)striated duct cell of exocrine glands gall bladder epithelial cell brushborder cell of proximal tubule of kidney distal tubule cell of kidneynonciliated cell of ductulus efferens epididymal principal cellepididymal basal cell Cells Specialized for Metabolism and Storagehepatocyte (liver cell) fat cells white fat brown fat lipocyte of liverEpithelial Cells Serving Primarily a Barrier Function, Lining the Lung,Gut, Exocrine Glands, and Urogenital Tract type I pneumocyte (lining airspace of lung) pancreatic duct cell (centroacinar cell) nonstriated ductcell of sweat gland, salivary gland, mammary gland parietal cell ofkidney glomerulus podocyte of kidney glomerulus cell of thin segment ofloop of Henle (in kidney) collecting duct cell (in kidney) duct cell ofseminal vesicle, prostate gland Epithelial Cells Lining Closed InternalBody Cavities vascular endothelial cells of blood vessels and lymphaticsfenestrated continuous splenic synovial cell (lining joint cavities,secreting largely hyaluronic acid) serosal cell (lining peritoneal,pleural, and pericardial cavities) squamous cell lining perilymphaticspace of ear cells lining endolymphatic space of ear squamous cellcolumnar cells of endolymphatic sac with microvilli without microvilli“dark” cell vestibular membrane cell (resembling choroid plexus cell)stria vascularis basal cell stria vascularis marginal cell cell ofClaudius cell of Boettcher choroid plexus cell (secreting cerebrospinalfluid) squamous cell of pia-arachnoid cells of ciliary epithelium of eyepigmented nonpigmented corneal “endothelial” cell Ciliated Cells withPropulsive Function of respiratory tract of oviduct and of endometriumof uterus (in female) of rete testis and ductulus efferens (in male) ofcentral nervous system (ependymal cell lining brain cavities) CellsSpecialized for Secretion of Extracellular Matrix epithelial: ameloblast(secreting enamel of tooth) planum semilunatum cell of vestibularapparatus of ear (secreting proteoglycan) interdental cell of organ ofCorti (secreting tectorial “membrane” covering hair cells of organ ofCorti) nonepithelial (connective tissue) fibroblasts (various-of looseconnective tissue, of cornea, of tendon, of reticular tissue of bonemarrow, etc.) pericyte of blood capillary nucleus pulposus cell ofintervertebral disc cementoblast/cementocyte (secreting bonelikecementum of root of tooth) odontoblast/odontocyte (secreting dentin oftooth) chondrocytes of hyaline cartilage, of fibrocartilage, of elasticcartilage osteoblast/osteocyte osteoprogenitor cell (stem cell ofosteoblasts) hyalocyte of vitreous body of eye stellate cell ofperilymphatic space of ear Contractile Cells skeletal muscle cells red(slow) white (fast) intermediate muscle spindleXXnuclear bag musclespindleXXnuclear chain satellite cell (stem cell) heart muscle cellsordinary nodal Purkinje fiber smooth muscle cells myoepithelial cells ofiris of exocrine glands Cells of Blood and Immune System red blood cellmegakaryocyte macrophages monocyte connective tissue macrophage(various) Langerhans cell (in epidermis) osteoclast (in bone) dendriticcell (in lymphoid tissues) microglial cell (in central nervous system)neutrophil eosinophil basophil mast cell T lymphocyte helper T cellsuppressor T cell killer T cell B lymphocyte IgM IgG IgA IgE killer cellstem cells for the blood and immune system (various) Sensory Transducersphotoreceptors rod cones blue sensitive green sensitive red sensitivehearing inner hair cell of organ of Corti outer hair cell of organ ofCorti acceleration and gravity type I hair cell of vestibular apparatusof ear type II hair cell of vestibular apparatus of ear taste type 11taste bud cell smell olfactory neuron basal cell of olfactory epithelium(stem cell for olfactory neurons) blood Ph carotid body cell type I typeII touch Merkel cell of epidermis primary sensory neurons specializedfor touch temperature primary sensory neurons specialized fortemperature cold sensitive heat sensitive pain primary sensory neuronsspecialized for pain configurations and forces in musculoskeletal systemproprioceptive primary sensory neurons Autonomic Neurons cholinergicadrenergic peptidergic Supporting Cells of Sense Organs and ofPeripheral Neurons supporting cells of organ of Corti inner pillar cellouter pillar cell inner phalangeal cell outer phalangeal cell bordercell Hensen cell supporting cell of vestibular apparatus supporting cellof taste bud (type I taste bud cell) supporting cell of olfactoryepithelium Schwann cell satellite cell (encapsulating peripheral nervecell bodies) enteric glial cell Neurons and Glial Cells of CentralNervous System neurons glial cells astrocyte oligodendrocyte Lens Cellsanterior lens epithelial cell lens fiber (crystallin-containing cell)Pigment Cells melanocyte, retinal pigmented epithelial cell Germ Cellsoogonium/oocyte spermatocyte spermatogonium (stem cell for spermatocyte)Nurse Cells ovarian follicle cell Sertoli cell (in testis) thymusepithelial cell Stem Cells embryonic stem cell embryonic germ cell adultstem cell fetal stem cell

[0410] IX. Diagnostic Assays

[0411] A) Introduction

[0412] 1) TRT Assays

[0413] The present invention provides a wide variety of assays for TRT,preferably hTRT, and telomerase. These assays provide, inter alia, thebasis for sensitive, inexpensive, convenient, and widely applicableassays for diagnosis and prognosis of a number of human diseases, ofwhich cancer is an illustrative example. As noted supra, hTRT geneproducts (protein and mRNA) are usually elevated in immortal human cellsrelative to most normal mortal cells (i.e., telomerase-negative cellsand most telomerase-positive normal adult somatic cells). Thus, in oneaspect, the invention provides assays useful for detecting or measuringthe presence, absence, or quantity of an hTRT gene product in a samplefrom, or containing, human or other mammalian or eukayotic cells tocharacterize the cells as immortal (such as a malignant tumor cell) ormortal (such as most normal somatic cells in adults) or as telomerasepositive or negative.

[0414] Any condition characterized by the presence or absence of an hTRTgene product (i.e., protein or RNA) may be diagnosed using the methodsand materials described herein. These include, as described more fullybelow, cancers, other diseases of accelerated cell proliferation,immunological disorders, fertility, infertility, and others. Moreover,because the degree to which telomerase activity is elevated in cancercells is correlated with characteristics of the tumor, such asmetastatic potential, monitoring hTRT, mRNA or protein levels can beused to estimate and predict the likely future progression of a tumor.

[0415] In one aspect, the diagnostic and prognostic methods of theinvention entail determining whether a human TRT gene product is presentin a biological sample (e.g., from a patient). In a second aspect, theabundance of hTRT gene product in a biological sample (e.g., from apatient) is determined and compared to the abundance in a control sample(e.g., normal cells or tissues). In a third aspect, the cellular orintracellular localization of an hTRT gene product is determined in acell or tissue sample. In a fourth aspect, host (e.g., patient) cellsare assayed to identify nucleic acids with sequences characteristic of aheritable propensity for abnormal hTRT gene expression (abnormalquantity, regulation, or product), such as is useful in geneticscreening or genetic counseling. In a fifth aspect, the assays of theinvention are used detect the presence of anti-hTRT antibodies (e.g., inpatient serum). The methods described below in some detail areindicative of useful assays that can be carried out using the sequencesand relationships disclosed herein. However, numerous variations orother applications of these assays will be apparent to those of ordinaryskill in the art in view of this disclosure.

[0416] It will be recognized that, although the assays below arepresented in terms of diagnostic and prognostic methods, they may beused whenever an hTRT gene, gene product, or variant is to be detected,quantified, or characterized. Thus, for example, the “diagnostic”methods described infra are useful for assays of hTRT or telomeraseduring production and purification of hTRT or human telomerase, forcharacterization of cell lines derived from human cells (e.g., toidentify immortal lines), for characterization of cells, non-humananimals, plants, fungi, bacteria or other organisms that comprise ahuman TRT gene or gene product (or fragments thereof).

[0417] As used herein, the term “diagnostic” has its usual meaning ofidentifying the presence or nature of a disease (e.g., cancer),condition (e.g., infertile, activated), or status (e.g., fertile), andthe term “prognostic” has its usual meaning of predicting the probabledevelopment and/or outcome of a disease or condition. Although these twoterms are used in somewhat different ways in a clinical setting, it willbe understood that any of the assays or assay formats disclosed below inreference to “diagnosis” are equally suitable for determination ofprognosis because it is well established that higher telomerase activitylevels are associated with poorer prognoses for cancer patients, andbecause the present invention provides detection methods specific forhTRT, which is expressed at levels that closely correlate withtelomerase activity in a cell.

[0418] 2) Diagnosis and Prognosis of Cancer

[0419] The determination of an hTRT gene, mRNA or protein level abovenormal or standard range is indicative of the presence oftelomerase-positive cells, or immortal, of which certain tumor cells areexamples. Because certain embryonic and fetal cells, as well as certainadult stem cells, express telomerase, the present invention alsoprovides methods for determining other conditions, such as pregnancy, bythe detection or isolation of telomerase positive fetal cells frommaternal blood. These values can be used to make, or aid in making, adiagnosis, even when the cells would not have been classified ascancerous or otherwise detected or classified using traditional methods.Thus, the methods of the present invention permit detection orverification of cancerous or other conditions associated with telomerasewith increased confidence, and at least in some instances at an earlierstage. The assays of the invention allow discrimination betweendifferent classes and grades of human tumors or other cell-proliferativediseases by providing quantitative assays for the hTRT gene and geneproducts and thereby facilitate the selection of appropriate treatmentregimens and accurate diagnoses. Moreover, because levels of telomeraseactivity can be used to distinguish between benign and malignant tumors(e.g., U.S. Pat. No. 5,489,508; Hiyama et al., 1997, Proc. Am Ass.Cancer Res. 38:637), to predict immanence of invasion (e.g., U.S. Pat.No. 5,639,613; Yashima et al., 1997, Proc. Am Ass. Cancer Res. 38:326),and to correlate with metastatic potential (e.g., U.S. Pat. No.5,648,215; Pandita et al, 1996, Proc. Am Ass. Cancer Res. 37:559), theseassays will be useful for prophylaxis, detection, and treatment of awide variety of human cancers.

[0420] For prognosis of cancers (or other diseases or conditionscharacterized by elevated telomerase), a prognostic value of hTRT geneproduct (mRNA or protein) or activity for a particular tumor type, classor grade, is determined as described infra. hTRT protein or mRNA levelsor telomerase activity in a patient can also be determined (e.g., usingthe assays disclosed herein) and compared to the prognostic level.

[0421] Depending on the assay used, in some cases the abundance of anhTRT gene product in a sample will be considered elevated whenever it isdetectable by the assay. Due to the low abundance of hTRT mRNA andprotein even in telomerase-positive cells, and the rarity ornon-existence of these gene products in normal or telomerase-negativecells, sensitive assays are required to detect the hTRT gene product ifpresent at all in normal cells. If less sensitive assays are selected,hTRT gene products will be undetectable in healthy tissue but will bedetectable in telomerase-positive cancer or other telomerase-positivecells. Typically, the amount of hTRT gene product in an elevated sampleis at least about five, frequently at least about ten, more often atleast about 50, and very often at least about 100 to 1000 times higherthan the levels in telomerase-negative control cells or cells fromhealthy tissues in an adult, where the percentage of telomerase-positivenormal cells is very low.

[0422] The diagnostic and prognostic methods of the present inventioncan be employed with any cell or tissue type of any origin and can beused to detect an immortal or neoplastic cell, or tumor tissue, orcancer, of any origin. Types of cancer that may be detected include, butare not limited to, all those listed supra in the discussion oftherapeutic applications of hTRT.

[0423] The assays of the invention are also useful for monitoring theefficacy of therapeutic intervention in patients being treated withanticancer regimens. Anticancer regimens that can be monitored includeall presently approved treatments (including chemotherapy, radiationtherapy, and surgery) and also includes treatments to be approved in thefuture, such as telomerase inhibition or activation therapies asdescribed herein. (See, e.g., See PCT Publication Nos. 96/01835 and96/40868 and U.S. Pat. No. 5,583,016; all of which are incorporated byreference in their entirety).

[0424] In another aspect, the assays described below are useful fordetecting certain variations in hTRT gene sequence (mutations andheritable hTRT alleles) that are indicative of a predilection forcancers or other conditions associated with abnormal regulation oftelomerase activity (infertility, premature aging).

[0425] 3) Diagnosis of Conditions Other than Cancer

[0426] In addition to diagnosis of cancers, the assays of the presentinvention have numerous other applications. The present inventionprovides reagents and methods/diagnosis of conditions or diseasescharacterized by under- or over-expression of telomerase or hTRT geneproducts in cells. In adults, a low level of telomerase activity isnormally found in a limited complement of normal human somatic cells,e.g., stem cells, activated lymphocytes and germ cells, and is absentfrom other somatic cells. Thus, the detection of hTRT or telomeraseactivity in cells in which it is normally absent or inactive, ordetection at abnormal (i.e., higher or lower than normal) levels incells in which hTRT is normally present at a low level (such as stemcells, activated lymphocytes and germ cells), can be diagnostic of atelomerase-related disease or condition or used to identify or isolate aspecific cell type (i.e., to isolate stem cells). Examples of suchdiseases and conditions include: diseases of cell proliferation,immunological disorders, infertility, diseases of immune cell function,pregnancy, fetal abnormalities, premature aging, and others. Moreover,the assays of the invention are useful for monitoring the effectivenessof therapeutic intervention (including but not limited to drugs thatmodulate telomerase activity) in a patient or in a cell- or animal-basedassay.

[0427] In one aspect, the invention provides assays useful fordiagnosing infertility. Human germ cells (e.g., spermatogonia cells,their progenitors or descendants) are capable of indefiniteproliferation and characterized by high telomerase activity. Abnormallevels or products or diminished levels of hTRT gene products can resultin inadequate or abnormal production of spermatozoa, leading toinfertility or disorders of reproduction. Accordingly, the inventionprovides assays (methods and reagents) for diagnosis and treatment of“telomerase-based” reproductive disorders. Similarly, the assays can beused to monitor the efficacy of contraceptives (e.g., malecontraceptives) that target or indirectly affect sperm production (andwhich would reduce hTRT levels or telomerase activity).

[0428] In another aspect, the invention provides assays for analysis oftelomerase and hTRT levels and function in stem cells, fetal cells,embryonic cells, activated lymphocytes and hematopoietic stem cells. Forexample, assays for hTRT gene product detection can be used to monitorimmune function generally (e.g., by monitoring the prevalence ofactivated lymphocytes or abundance of progenitor stem cells), toidentify or select or isolate activated lymphocytes or stem cells (basedon elevated hTRT levels), and to monitor the efficacy of therapeuticinterventions targeting these tissues (e.g., immunosuppressive agents ortherapeutic attempt to expand a stem cell population).

[0429] The invention also provides assays useful for identification ofanti-telomerase and anti-TRT immunoglobulins (found in serum from apatient). The materials and assays described herein can be used toidentify patients in which such autoimmune antibodies are found,permitting diagnosis and treatment of the condition associated with theimmunoglobulins.

[0430] 4) Monitoring Cells in Culture

[0431] The assays described herein are also useful for monitoring theexpression of hTRT gene products and characterization of hTRT genes incells ex vivo or in vitro. Because elevated hTRT levels arecharacteristic of immortalized cells, the assays of the invention can beused, for example, to screen for, or identify, immortalized cells or toidentify an agent capable of mortalizing immortalized cells byinhibiting hTRT expression or function. For example, the assay will beuseful for identifying cells immortalized by increased expression ofhTRT in the cell, e.g., by the expression of a recombinant hTRT or byincreased expression of an endogenously coded hTRT (e.g., by promoteractivation).

[0432] Similarly, these assays may be used to monitor hTRT expression intransgenic animals or cells (e.g., yeast or human cells containing anhTRT gene). In particular, the effects of certain treatments (e.g.,application of known or putative telomerase antagonists) on the hTRTlevels in human and nonhuman cells expressing the hTRT of the inventioncan be used for identifying useful drugs and drug candidates (e.g.,telomerase activity-modulating drugs).

[0433] B) Normal, Diagnostic, and Prognostic Values

[0434] Assays for the presence or quantity of hTRT gene products may becarried out and the results interpreted in a variety of ways, dependingon the assay format, the nature of the sample being assayed, and theinformation sought. For example, the steady state abundance of hTRT geneproducts is so low in most human somatic tissues that they areundetectable by certain assays. Moreover, there is generally notelomerase activity in the cells of these tissues, making verificationof activity quite easy. Conversely, hTRT protein and/or hTRT mRNA ortelomerase is sufficiently abundant in other telomerase-positivetissues, e.g., malignant tumors, so that the same can be detected usingthe same assays. Even in those somatic cell types in which low levels oftelomerase activity can normally be detected (e.g., stem cells, andcertain activated hematopoietic system cells), the levels of hTRT mRNAand telomerase activity are a small fraction (e.g., estimated at about1% or less) of the levels in immortal cells; thus, immortal and mortalcells may be easily distinguished by the methods of the presentinvention. It will be appreciated that, when a “less sensitive” assay isused, the mere detection of the hTRT gene product in a biological samplecan itself be diagnostic, without the requirement for additionalanalysis. Moreover, although the assays described below can be madeexquisitely sensitive, they may also, if desired, be made less sensitive(e.g., through judicious choice of buffers, wash conditions, numbers ofrounds of amplification, reagents, and/or choice of signal amplifiers).Thus, virtually any assay can be designed so that it detects hTRT geneproducts only in biological samples in which they are present at aparticular concentration, e.g. a higher concentration than in healthy orother control tissue. In this case, any detectable level of hTRT mRNA orprotein will be considered elevated in cells from post-natal humansomatic tissue (other than hematopoietic cells and other stem cells).

[0435] In some cases, however, it will be desirable to establish normalor baseline values (or ranges) for hTRT gene product expression levels,particularly when very sensitive assays capable of detecting very lowlevels of hTRT gene products that may be present in normal somatic cellsare used. Normal levels of expression or normal expression products canbe determined for any particular population, subpopulation, or group oforganisms according to standard methods well known to those of skill inthe art and employing the methods and reagents of the invention.Generally, baseline (normal) levels of hTRT protein or hTRT mRNA aredetermined by quantitating the amount of hTRT protein and/or mRNA inbiological samples (e.g., fluids, cells or tissues) obtained from normal(healthy) subjects, e.g., a human subject. For certain samples andpurposes, one may desire to quantitate the amount of hTRT gene producton a per cell, or per tumor cell, basis. To determine the cellularity ofa sample, one may measure the level of a constitutively expressed geneproduct or other gene product expressed at known levels in cells of thetype from which the sample was taken. Alternatively, normal values ofhTRT protein or hTRT mRNA can be determined by quantitating the amountof hTRT protein/RNA in cells or tissues known to be healthy, which areobtained from the same patient from whom diseased (or possibly diseased)cells are collected or from a healthy individual. Alternatively,baseline levels can be defined in some cases as the level present innon-immortal human somatic cells in culture. It is possible that normal(baseline) values may differ somewhat between different cell types (forexample, hTRT mRNA levels will be higher in testis than kidney), oraccording to the age, sex, or physical condition of a patient. Thus, forexample, when an assay is used to determine changes in hTRT levelsassociated with cancer, the cells used to determine the normal range ofhTRT gene product expression can be cells from persons of the same or adifferent age, depending on the nature of the inquiry. Application ofstandard statistical methods used in molecular genetics permitsdetermination of baseline levels of expression, as well as permitsidentification of significant deviations from such baseline levels.

[0436] In carrying out the diagnostic and prognostic methods of theinvention, as described above, it will sometimes be useful to refer to“diagnostic” and “prognostic values” As used herein, “diagnostic value”refers to a value that is determined for the hTRT gene product detectedin a sample which, when compared to a normal (or “baseline”) range ofthe hTRT gene product is indicative of the presence of a disease. Thedisease may be characterized by high telomerase activity (e.g., cancer),the absence of telomerase activity (e.g., infertility), or someintermediate value. “Prognostic value” refers to an amount of the hTRTgene product detected in a given cell type (e.g., malignant tumor cell)that is consistent with a particular diagnosis and prognosis for thedisease (e.g., cancer). The amount (including a zero amount) of the hTRTgene product detected in a sample is compared to the prognostic valuefor the cell such that the relative comparison of the values indicatesthe presence of disease or the likely outcome of the disease (e.g.,cancer) progression. In one embodiment, for example, to assess tumorprognosis, data are collected to obtain a statistically significantcorrelation of hTRT levels with different tumor classes or grades. Apredetermined range of hTRT levels is established for the same cell ortissue sample obtained from subjects having known clinical outcomes. Asufficient number of measurements is made to produce a statisticallysignificant value (or range of values) to which a comparison will bemade. The predetermined range of hTRT levels or activity for a givencell or tissue sample can then be used to determine a value or range forthe level of hTRT gene product that would correlate to favorable (orless unfavorable) prognosis (e.g., a “low level” in the case of cancer).A range corresponding to a “high level” correlated to an (or a more)unfavorable prognosis in the case of cancer can similarly be determined.The level of hTRT gene product from a biological sample (e.g., a patientsample) can then be determined and compared to the low and high rangesand used to predict a clinical outcome.

[0437] Although the discussion above refers to cancer for illustration,it will be understood that diagnostic and prognostic values can also bedetermined for other diseases (e.g., diseases of cell proliferation) andconditions and that, for diseases or conditions other than cancer, a“high” level may be correlated with the desired outcome and a “low”level correlated with an unfavorable outcome. For example, some diseasesmay be characterized by a deficiency (e.g., low level) of telomeraseactivity in stem cells, activated lymphocytes, or germline cells. Insuch cases, “high” levels of hTRT gene products relative to cells ofsimilar age and/or type (e.g., from other patients or other tissues in aparticular patient) may be correlated with a favorable outcome.

[0438] It will be appreciated that the assay methods do not necessarilyrequire measurement of absolute values of hTRT, unless it is so desired,because relative values are sufficient for many applications of themethods of the present invention. Where quantitation is desirable, thepresent invention provides reagents such that virtually any known methodfor quantitating gene products can be used.

[0439] The assays of the invention may also be used to evaluate theefficacy of a particular therapeutic treatment regime in animal studies,in clinical trials, or in monitoring the treatment of an individualpatient. In these cases, it may be desirable to establish the baselinefor the patient prior to commencing therapy and to repeat the assays oneor more times through the course of treatment, usually on a regularbasis, to evaluate whether hTRT levels are moving toward the desiredendpoint (e.g., reduced expression of hTRT when the assay is for cancer)as a result of the treatment.

[0440] One of skill will appreciate that, in addition to the quantity orabundance of hTRT gene products, variant or abnormal expression patterns(e.g., abnormal amounts of RNA splicing variants) or variant or abnormalexpression products (e.g., mutated transcripts, truncated or non-sensepolypeptides) may also be identified by comparison to normal expressionlevels and normal expression products. In these cases determination of“normal” or “baseline” involves identifying healthy organisms and/ortissues (i.e. organisms and/or tissues without hTRT expressiondisregulation or neoplastic growth) and measuring expression levels ofthe variant hTRT gene products (e.g., splicing variants), or sequencingor detecting the hTRT gene, mRNA, or reverse transcribed cDNA to obtainor detect typical (normal) sequence variations. Application of standardstatistical methods used in molecular genetics permits determination ofsignificant deviations from such baseline levels.

[0441] C) Detection and Quantitation of TRT Gene Products

[0442] As has been emphasized herein, hTRT gene products are usuallyfound in most normal somatic cells at extremely low levels. For example,the mRNA encoding hTRT protein is extremely rare or absent in alltelomerase-negative cell types studied thus far. In immortal cells, suchas 293 cells, hTRT mRNA may be present at only about 100 copies percell, while normal somatic cells may have as few as one or zero copiesper cell. It will thus be apparent that, when highly sensitive assaysfor hTRT gene products are desired, it will sometimes be advantageous toincorporate signal or target amplification technologies into the assayformat. See, for example, Plenat et al., 1997, Ann. Pathol. 17:17(fluoresceinyl-tyramide signal amplification); Zehbe et al., 1997, J.Pathol. 150:1553 (catalyzed reporter deposition); other referenceslisted herein (e.g., for bDNA signal amplification, for PCR and othertarget amplification formats); and other techniques known in the art.

[0443] As noted above, it is often unnecessary to quantitate the hTRTmRNA or protein in the assays disclosed herein, because the detection ofan hTRT gene product (under assay conditions in which the product is notdetectable in control, e.g., telomerase-negative cells) is in itselfsufficient for a diagnosis. As another example, when the levels ofproduct found in a test (e.g., tumor) and control (e.g., healthy cell)samples are directly compared, quantitation may be superfluous.

[0444] When desired, however, quantities of hTRT gene product measuredin the assays described herein may be described in a variety of ways,depending on the method of measurement and convenience. Thus, normal,diagnostic, prognostic, high or low quantities of hTRT protein/mRNA maybe expressed as standard units of weight per quantity of biologicalsample (e.g., picograms per gram tissue, picograms per 10¹² cells), as anumber of molecules per quantity of biological sample (e.g.,transcripts/cell, moles/cell), as units of activity per cell or perother unit quantity, or by similar methods. The quantity of hTRT geneproduct can also be expressed in relation to the quantity of anothermolecule; examples include: number of hTRT transcripts in sample/numberof 28S rRNA transcripts in sample; nanograms of hTRT protein/nanogramsof total protein; and the like.

[0445] When measuring hTRT gene products in two (or more) differentsamples, it will sometimes be useful to have a common basis ofcomparison for the two samples. For example, when comparing a sample ofnormal tissue and a sample of cancerous tissue, equal amounts of tissue(by weight, volume, number of cells, etc.) can be compared.Alternatively, equivalents of a marker molecule (e.g., 28S rRNA, hTR,telomerase activity, telomere length, actin) may be used. For example,the amount of hTRT protein in a healthy tissue sample containing 10picograms of 28S rRNA can be compared to a sample of diseased tissuecontaining the same amount of 28S rRNA.

[0446] It will also be recognized by those of skill that virtually anyof the assays described herein can be designed to be quantitative.Typically, a known quantity or source of an hTRT gene product (e.g.,produced using the methods and compositions of the invention) is used tocalibrate the assay.

[0447] In certain embodiments, assay formats are chosen that detect thepresence, absence, or abundance of an hTRT allele or gene product ineach cell in a sample (or in a representative sampling). Examples ofsuch formats include those that detect a signal by histology (e.g.,immunohistochemistry with signal-enhancing or target-enhancingamplification steps) or fluorescence-activated cell analysis or cellsorting (FACS). These formats are particularly advantageous when dealingwith a highly heterogeneous cell population (e.g., containing multiplecells types in which only one or a few types have elevated hTRT levels,or a population of similar cells expressing telomerase at differentlevels).

[0448] D) Sample Collection

[0449] The hTRT gene or gene product (i.e., mRNA or polypeptide) ispreferably detected and/or quantified in a biological sample. Suchsamples include, but are not limited to, cells (including whole cells,cell fractions, cell extracts, and cultured cells or cell lines),tissues (including blood, blood cells (e.g., white cells), and tissuesamples such as fine needle biopsy samples (e.g., from prostate, breast,thyroid, etc.)), body fluids (e.g., urine, sputum, amniotic fluid,blood, peritoneal fluid, pleural fluid, semen) or cells collectedtherefrom (e.g., bladder cells from urine, lymphocytes from blood),media (from cultured cells or cell lines), and washes (e.g., of bladderand lung). Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes. For cancer diagnosisand prognosis, a sample will be obtained from a cancerous orprecancerous or suspected cancerous tissue or tumor. It will sometimesbe desirable to freeze a biological sample for later analysis (e.g.,when monitoring efficacy of drug treatments).

[0450] In some cases, the cells or tissues may be fractionated beforeanalysis. For example, in a tissue biopsy from a patient, a cell sorter(e.g., a fluorescence-activated cell sorter) may be used to sort cellsaccording to characteristics such as expression of a surface antigen(e.g., a tumor specific antigen) according to well known methods.

[0451] Although the sample is typically taken from a human patient orcell line, the assays can be used to detect hTRT homolog genes or geneproducts in samples from other animals. Alternatively, hTRT genes andgene products can be assayed in transgenic animals or organismsexpressing a human TRT protein or nucleic acid sequence.

[0452] The sample may be pretreated as necessary by dilution in anappropriate buffer solution or concentrated, if desired. Any of a numberof standard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris-buffer, or the like, at physiologicalpH can be used.

[0453] A “biological sample” obtained from a patient can be referred toeither as a “biological sample” or a “patient sample.” It will beappreciated that analysis of a “patient sample” need not necessarilyrequire removal of cells or tissue from the patient. For example,appropriately labeled hTRT-binding agents (e.g., antibodies or nucleicacids) can be injected into a patient and visualized (when bound to thetarget) using standard imaging technology (e.g., CAT, NMR, and thelike.)

[0454] E) Nucleic Acid Assays

[0455] In one embodiment, this invention provides for methods ofdetecting and/or quantifying expression of hTRT mRNAs (includingsplicing or sequence variants and alternative alleles). In analternative embodiment, the invention provides methods for detecting andanalyzing normal or abnormal hTRT genes (or fragments thereof). The formof such qualitative or quantitative assays may include, but is notlimited to, amplification-based assays with or without signalamplification, hybridization based assays, and combinationamplification-hybridization assays. It will be appreciated by those ofskill that the distinction between hybridization and amplification isfor convenience only: as illustrated in the examples below, many assayformats involve elements of both hybridization and amplification, sothat the categorization is somewhat arbitrary in some cases.

[0456] 1) Preparation of Nucleic Acids

[0457] In some embodiments, nucleic acid assays are performed with asample of nucleic acid isolated from the cell, tissue, organism, or cellline to be tested. The nucleic acid (e.g., genomic DNA, RNA or cDNA) maybe “isolated” from the sample according to any of a number of methodswell known to those of skill in the art. In this context, “isolated”refers to any separation of the species or target to be detected fromany other substance in the mixture, but does not necessarily indicate asignificant degree of purification of the target. One of skill willappreciate that, where alterations in the copy number of the hTRT geneare to be detected, genomic DNA is the target to be detected.Conversely, where expression levels of a gene or genes are to bedetected, RNA is the target to be detected in a nucleic acid-basedassay. In one preferred embodiment, the nucleic acid sample is the totalmRNA (i.e., poly(A)⁺ RNA) in a biological sample. Methods for isolatingnucleic acids are well known to those of skill in the art and aredescribed, for example, Tijssen, P. ed. of LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACIDPROBES, PART I. THEORY AND NUCLEIC ACID PREPARATION, Elsevier, N.Y.(1993) Chapt. 3, which is incorporated herein by reference. In oneembodiment, the total nucleic acid is isolated from a given sample usingan acid guanidinium-phenol-chloroform extraction method and poly(A)+mRNA is isolated by oligo-dT column chromatography or by using (dT)_(n)magnetic beads (see, e.g., Sambrook et al., and Ausubel et al., supra).

[0458] In alternative embodiments, it is not necessary to isolatenucleic acids (e.g., total or polyA⁺ RNA) from the biological sampleprior to carrying out amplification, hybridization or other assays.These embodiments have certain advantages when hTRT RNA is to bemeasured, because they reduce the possibility of loss of hTRT mRNAduring isolation and handling. For example, many amplificationtechniques such as PCR and RT-PCR defined above can be carried out usingpermeabilized cells (histological specimens and FACS analyses), wholelysed cells, or crude cell fractions such as certain cell extracts.Preferably, steps are taken to preserve the integrity of the targetnucleic acid (e.g., mRNA) if necessary (e.g., addition of RNAaseinhibitors). Amplification and hybridization assays can also be carriedout in situ, for example, in thin tissue sections from a biopsy sampleor from a cell monolayer (e.g., blood cells or disagregated tissueculture cells). Amplification can also be carried out in an intact wholecell or fixed cells. For example, PCR, RT-PCR, or LCR amplificationmethods may be carrier out, as is well known in the art, in situ, e.g.,using a polymerase or ligase, a primer or primer(s), and(deoxy)ribonucleoside triphosphates (if a polymerase is employed), andreverse transcriptase and primer (if RNA is to be transcribed and thecDNA is to be detected) on fixed, permeabilized, or microinjected cellsto amplify target hTRT RNA or DNA. Cells containing hTRT RNA (e.g.,telomerase positive cells) or an hTRT DNA sequence of interest can thenbe detected. This method is often useful when fluorescently-labeleddNTPs, primers, or other components are used in conjunction withmicroscopy, FACS analysis or the equivalent.

[0459] 2) Amplification Based Assays

[0460] In one embodiment, the assays of the present invention areamplification-based assays for detection of an hTRT gene or geneproduct. In an amplification based assay, all or part of an hTRT gene ortranscript (e.g., mRNA or cDNA; hereinafter also referred to as“target”) is amplified, and the amplification product is then detecteddirectly or indirectly. When there is no underlying gene or gene productto act as a template, no amplification product is produced (e.g., of theexpected size), or amplification is non-specific and typically there isno single amplification product. In contrast, when the underlying geneor gene product is present, the target sequence is amplified, providingan indication of the presence and/or quantity of the underlying gene ormRNA. Target amplification-based assays are well known to those of skillin the art.

[0461] The present invention provides a wide variety of primers andprobes for detecting hTRT genes and gene products. Such primers andprobes are sufficiently complementary to the hTRT gene or gene productto hybridize to the target nucleic acid. Primers are typically at least6 bases in length, usually between about 10 and about 100 bases,typically between about 12 and about 50 bases, and often between about14 and about 25 bases in length. One of skill, having reviewed thepresent disclosure, will be able, using routine methods, to selectprimers to amplify all, or any portion, of the hTRT gene or geneproduct, or to distinguish between variant gene products, hTRT alleles,and the like. Table 2 lists illustrative primers useful for PCRamplification of the hTRT, or specific hTRT gene products or regions. Asis known in the art, single oligomers (e.g., U.S. Pat. No. 5,545,522),nested sets of oligomers, or even a degenerate pool of oligomers may beemployed for amplification, e.g., as illustrated by the amplification ofthe Tetrahymena TRT cDNA as described infra.

[0462] The invention provides a variety of methods for amplifying anddetecting an hTRT gene or gene product, including the polymerase chainreaction (including all variants, e.g., reverse-transcriptase-PCR; theSunrise Amplification System (Oncor, Inc, Gaithersburg Md.); andnumerous others known in the art). In one illustrative embodiment, PCRamplification is carried out in a 50 μl solution containing the nucleicacid sample (e.g., cDNA obtained through reverse transcription of hTRTRNA), 100 μM in each dNTP (dATP, dCTP, dGTP and dTTP; Pharmacia LKBBiotechnology, NJ), the hTRT-specific PCR primer(s), 1 unit/Taqpolymerase (Perkin Elmer, Norwalk Conn.), 1×PCR buffer (50 mM KCl, 10 mMTris, pH 8.3 at room temperature, 1.5 mM MgCl₂, 0.01% gelatin) with theamplification run for about 30 cycles at 94° for 45 sec, 55° for 45 secand 72° for 90 sec. However, as will be appreciated, numerous variationsmay be made to optimize the PCR amplification for any particularreaction.

[0463] Other suitable target amplification methods include the ligasechain reaction (LCR; e.g., Wu and Wallace, 1989, Genomics 4:560;Landegren et al., 1988, Science, 241: 1077, Barany, 1991, Proc. Natl.Acad. Sci. USA 88:189 and Barringer et al., 1990, Gene, 89: 117); stranddisplacement amplification (SDA; e.g., Walker et al., 1992, Proc. Natl.Acad. Sci. U.S.A. 89:392-396); transcription amplification (e.g., Kwohet al., 1989, Proc. Natl. Acad. Sci. USA, 86: 1173); self-sustainedsequence replication (3 SR; e.g., Fahy et al., 1992, PCR Methods Appl.1:25, Guatelli et al., 1990, Proc. Nat. Acad. Sci. USA, 87: 1874); thenucleic acid sequence based amplification (NASBA, Cangene, Mississauga,Ontario; e.g., Compton, 1991, Nature 350:91); the transcription-basedamplification system (TAS); and the self-sustained sequence replicationsystem (SSR). Each of the aforementioned publications is incorporatedherein by reference. One useful variant of PCR is PCR ELISA (e.g.,Boehringer Mannheim Cat. No. 1 636 111) in which digoxigenin-dUTP isincorporated into the PCR product. The PCR reaction mixture is denaturedand hybridized with a biotin-labeled oligonucleotide designed to annealto an internal sequence of the PCR product. The hybridization productsare immobilized on streptavidin coated plates and detected usinganti-digoxigenin antibodies. Examples of techniques sufficient to directpersons of skill through in vitro amplification methods are found in PCRTECHNOLOGY: PRINCIPLES AND APPLICATIONS FOR DNA AMPLIFICATION, H.Erlich, Ed. Freeman Press, New York, N.Y. (1992); PCR PROTOCOLS: A GUIDETO METHODS AND APPLICATIONS, eds. Innis, Gelfland, Snisky, and White,Academic Press, San Diego, Calif. (1990); Mattila et al., 1991, NucleicAcids Res. 19: 4967; Eckert and Kunkel, (1991) PCR METHODS ANDAPPLICATIONS 1: 17; PCR, eds. McPherson, Quirkes, and Taylor, IRL Press,Oxford; U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188; Barringer etal., 1990, Gene, 89:117; Lomell et al., 1989, J. Clin. Chem., 35:1826,each of which is incorporated herein for all purposes.

[0464] Amplified products may be directly analyzed, e.g., by size asdetermined by gel electrophoresis; by hybridization to a target nucleicacid immobilized on a solid support such as a bead, membrane, slide, orchip; by sequencing; immunologically, e.g., by PCR-ELISA, by detectionof a fluorescent, phosphorescent, or radioactive signal; or by any of avariety of other well-known means. For example, an illustrative exampleof a detection method uses PCR primers augmented with hairpin loopslinked to fluorescein and a benzoic acid derivative that serves as aquencher, such that fluorescence is emitted only when the primers unfoldto bind their targets and replication occurs.

[0465] Because hTRT mRNA is typically expressed as an extremely raretranscript, present at very low levels even in telomerase positivecells, it is often desirable to optimize or increase the signalresulting from the amplification step. One way to do this is to increasethe number of cycles of amplification. For example, although 20-25cycles are adequate for amplification of most mRNAs using the polymerasechain reaction under standard reaction conditions, detection of hTRTmRNA in many samples can require as many as 30 to 35 cycles ofamplification, depending on detection format and efficiency ofamplification. It will be recognized that judicious choice of theamplification conditions including the number of amplification cyclescan be used to design an assay that results in an amplification productonly when there is a threshold amount of target in the test sample(i.e., so that only samples with a high level of hTRT mRNA give a“positive” result). In addition, methods are known to increase signalproduced by amplification of the target sequence. Methods for augmentingthe ability to detect the amplified target include signal amplificationsystem such as: branched DNA signal amplification (e.g., U.S. Pat. No.5,124,246; Urdea, 1994, Bio/Tech. 12:926); tyramide signal amplification(TSA) system (Du Pont); catalytic signal amplification (CSA; Dako); QBeta Replicase systems (Tyagi et al., 1996, Proc. Nat. Acad. Sci. USA,93: 5395); or the like.

[0466] One of skill in the art will appreciate that whateveramplification method is used, a variety of quantitative methods known inthe art can be used if quantitation is desired. For example, whendesired, two or more polynucleotides can be co-amplified in a singlesample. This method can be used as a convenient method of quantitatingthe amount of hTRT mRNA in a sample, because the reverse transcriptionand amplification reactions are carried out in the same reaction for atarget and control polynucleotide. The co-amplification of the controlpolynucleotide (usually present at a known concentration or copy number)can be used for normalization to the cell number in the sample ascompared to the amount of hTRT in the sample. Suitable controlpolynucleotides for co-amplification reactions include DNA, RNAexpressed from housekeeping genes, constitutively expressed genes, andin vitro synthesized RNAs or DNAs added to the reaction mixture.Endogenous control polynucleotides are those that are already present inthe sample, while exogenous control polynucleotides are added to asample, creating a “spiked” reaction. Illustrative control RNAs includeβ-actin RNA, GAPDH RNA, snRNAs, hTR, and endogenously expressed 28S rRNA(see Khan et al., 1992, Neurosci. Lett. 147:114). Exogenous controlpolynucleotides include a synthetic AW106 cRNA, which may be synthesizedas a sense strand from pAW106 by T7 polymerase. It will be appreciatedthat for the co-amplification method to be useful for quantitation, thecontrol and target polynucleotides must typically both be amplified in alinear range. Detailed protocols for quantitative PCR may be found inPCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, Innis et al.,Academic Press, Inc. N.Y., (1990) and Ausubel et al., supra (Unit 15)and Diaco, R. (1995) Practical Considerationsfor the Design ofQuantitative PCR Assays, in PCR STRATEGIES, pg. 84-108, Innis et al.eds, Academic Press, New York.

[0467] Depending on the sequence of the endogenous or exogenousstandard, different primer sets may be used for the co-amplificationreaction. In one method, called competitive amplification, quantitativePCR involves simultaneously co-amplifying a known quantity of a controlsequence using the same primers used for amplification of the targetnucleic acid (one pair of 2 primers). In an alternative embodiment,known as non-competitive competition, the control sequence and thetarget sequence (e.g., hTRT cDNA) are amplified using different primers(i.e., 2 pairs of 2 primers). In another alternative embodiment, calledsemi-competitive amplification, three primers are used, one of which ishTRT-specific, one of which is control specific, and one of which iscapable of annealing to both the target and control sequences.Semi-competitive amplification is described in U.S. Pat. No. 5,629,154,which is incorporated herein by reference.

[0468] 3) Hybridization-Based Assays

[0469] a) Generally

[0470] A variety of methods for specific DNA and RNA measurement usingnucleic acid hybridization techniques are known to those of skill in theart (see Sambrook et al., supra). Hybridization based assays refer toassays in which a probe nucleic acid is hybridized to a target nucleicacid. Usually the nucleic acid hybridization probes of the invention areentirely or substantially identical to a contiguous sequence of the hTRTgene or RNA sequence. Preferably, nucleic acid probes are at least about10 bases, often at least about 20 bases, and sometimes at least about200 bases or more in length. Methods of selecting nucleic acid probesequences for use in nucleic acid hybridization are discussed inSambrook et al., supra. In some formats, at least one of the target andprobe is immobilized. The immobilized nucleic acid may be DNA, RNA, oranother oligo- or poly-nucleotide, and may comprise natural ornon-naturally occurring nucleotides, nucleotide analogs, or backbones.Such assays may be in any of several formats including: Southern,Northern, dot and slot blots, high-density polynucleotide oroligonucleotide arrays (e.g., GeneChips™ Affymetrix), dip sticks, pins,chips, or beads. All of these techniques are well known in the art andare the basis of many commercially available diagnostic kits.Hybridization techniques are generally described in Hames et al., ed.,NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH IRL Press, (1985); Galland Pardue Proc. Natl. Acad. Sci., U.S.A., 63: 378-383 (1969); and Johnet al., Nature, 223: 582-587 (1969).

[0471] A variety of nucleic acid hybridization formats are known tothose skilled in the art. For example, one common format is directhybridization, in which a target nucleic acid is hybridized to alabeled, complementary probe. Typically, labeled nucleic acids are usedfor hybridization, with the label providing the detectable signal. Onemethod for evaluating the presence, absence, or quantity of hTRT mRNA iscarrying out a Northern transfer of RNA from a sample and hybridizationof a labeled hTRT specific nucleic acid probe, as illustrated in Example2. As was noted supra, hTRT mRNA, when present at all, is present invery low quantities in most cells. Therefore, when Northernhybridization is used, it will often be desirable to use anamplification step (or, alternatively, large amounts of starting RNA). Auseful method for evaluating the presence, absence, or quantity of DNAencoding hTRT proteins in a sample involves a Southern transfer of DNAfrom a sample and hybridization of a labeled hTRT specific nucleic acidprobe.

[0472] Other common hybridization formats include sandwich assays andcompetition or displacement assays. Sandwich assays are commerciallyuseful hybridization assays for detecting or isolating nucleic acidsequences. Such assays utilize a “capture” nucleic acid covalentlyimmobilized to a solid support and a labeled “signal” nucleic acid insolution. The biological or clinical sample will provide the targetnucleic acid. The “capture” nucleic acid and “signal” nucleic acid probehybridize with the target nucleic acid to form a “sandwich”hybridization complex. To be effective, the signal nucleic acid cannothybridize with the capture nucleic acid.

[0473] b) Chip-Based and Slide-Based Assays

[0474] The present invention also provides probe-based hybridizationassays for hTRT gene products employing arrays of immobilizedoligonucleotide or polynucleotides to which an hTRT nucleic acid canhybridize (i.e., to some, but usually not all or even most, of theimmobilized oligo- or poly-nucleotides). High density oligonucleotidearrays or polynucleotide arrays provide a means for efficientlydetecting the presence and characteristics (e.g., sequence) of a targetnucleic acid (e.g., hTRT gene, mRNA, or cDNA). Techniques are known forproducing arrays containing thousands of oligonucleotides complementaryto defined sequences, at defined locations on a surface usingphotolithographic techniques for synthesis in situ (see, e.g., U.S. Pat.Nos. 5,578,832; 5,556,752; and 5,510,270; Fodor et al., 1991, Science251:767; Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91:5022; andLockhart et al., 1996, Nature Biotech 14:1675) or other methods forrapid synthesis and deposition of defined oligonucleotides (Blanchard etal., 1996, Biosensors & Bioelectronics 11:687). When these methods areused, oligonucleotides (e.g., 20-mers) of known sequence are synthesizeddirectly on a surface such as a derivatized glass slide. Usually, thearray produced is redundant, having several oligonucleotide probes onthe chip specific for the hTRT polynucleotide to be detected.

[0475] Combinations of oligonucleotide probes can be designed to detectalternatively spliced mRNAs, or to identify which of various hTRTalleles is expressed in a particular sample.

[0476] In one illustrative embodiment, cDNA prepared by reversetranscription of total RNA from a test cell is amplified (e.g., usingPCR). Typically the amplification product is labeled, e.g., byincorporation of a fluorescently labeled dNTP. The labeled cDNAs arethen hybridized to a chip comprising oligonucleotide probescomplementary to various subsequences of the hTRT gene. The positions ofhybridization are determined (e.g., in accordance with the generalmethods of Shalon et al., 1996, Genome Research 6:639 or Schena et al.,1996, Genome Res. 6:639), and sequence (or other information) deducedfrom the hybridization pattern, by means well known in the art.

[0477] In one embodiment, two cDNA samples, each labeled with adifferent fluorescent group, are hybridized to the same chip. The ratioof the hybridization of each labeled sample to sites complementary tothe hTRT gene are then assayed. If both samples contain the same amountof hTRT mRNA, the ratio of the two fluors will be 1:1 (it will beappreciated that the signal from the fluors may need to be adjusted toaccount for any difference in the molar sensitivity of the fluors). Incontrast, if one sample is from a healthy (or control) tissue and thesecond sample is from a cancerous tissue the fluor used in the secondsample will predominate.

[0478] c) In Situ Hybridization

[0479] An alternative means for detecting expression of a gene encodingan hTRT protein is in situ hybridization. In situ hybridization assaysare well known and are generally described in Angerer et al., METHODSENZYMOL., 152: 649-660 (1987) and Ausubel et al., supra. In an in situhybridization assay, cells or tissue specimens are fixed to a solidsupport, typically in a permeablilized state, typically on a glassslide. The cells are then contacted with a hybridization solution at amoderate temperature to permit annealing of labeled nucleic acid probes(e.g., ³⁵S-labeled riboprobes, fluorescently labeled probes) completelyor substantially complementary to hTRT. Free probe is removed by washingand/or nuclease digestion, and bound probe is visualized directly on theslide by autoradiography or an appropriate imaging techniques, as isknown in the art.

[0480] 4) Specific Detection of Variants

[0481] As noted supra and illustrated in the Examples (e.g., Example 9),amplification primers or probes can be selected to provide amplificationproducts that span specific deletions, truncations, and insertions,thereby facilitating the detection of specific variants or abnormalitiesin the hTRT mRNA.

[0482] One example of an hTRT variant gene product that may be detectedis an hTRT RNA such as a product (SEQ ID NO:4) described supra and inExample 9. The biological function, if any, of the Δ182 variant(s) isnot known; however, the truncated hTRT protein putatively encoded by thevariant may be involved in regulation of telomerase activity, e.g., byassembling a non-functional telomerase RNP that titrates telomerasecomponents. Alternatively, negative regulation of telomerase activitycould be accomplished by directing hTRT pre-mRNA (nascent mRNA)processing in a manner leading to elimination of the full length mRNAand reducing hTRT mRNA levels and increasing Δ182 hTRT RNA levels. Forthese and other reasons, the ability to detect Δ182 variants is useful.In addition, it will sometimes be desirable, in samples in which twospecies of hTRT RNA are present (such as a Δ182 hTRT RNA and hTRT RNAencoding the full-length hTRT protein) to compare their relative and/orabsolute abundance.

[0483] The invention provides a variety of methods for detection of Δ182variants. For example, amplification using primer pairs spanning the 182basepair deletion will result in different sized products correspondingto the deleted and undeleted hTRT RNAs, if both are present, which canbe distinguished on the basis of size (e.g., by gel electrophoresis).Examples of primer pairs useful for amplifying the region spanning the182 bp deletion include TCP1.14 and TCP1.15 (primer set 1), or TCP1.25and billTCP6 (primer set 2) (see Table 2). These primer pairs can beused individually or in a nested PCR experiment where primer set 1 isused first. It will also be apparent to one of skill that hybridizationmethods (e.g., Northern hybridization) or RNAse protection assays usingan hTRT nucleic acid probe of the invention can be used to detect anddistinguish hTRT RNA variants.

[0484] Another suitable method entails PCR amplification (or theequivalent) using three primers. Analogous to the semi-competitivequantitative PCR method described in greater detail supra, one primer isspecific to each of the hTRT RNA species (e.g., as illustrated in Table4) and one primer is complementary to both species (e.g., TCP1.25(2270-2288)). An example of a primer specific to SEQ ID NO:1 is one thatanneals within the 182 nucleotide sequence (i.e., nucleotides 2345 to2526 of SEQ ID NO:1), e.g., TCP1.73 (2465-2445). For example, a primerspecific to SEQ ID NO:4 (a Δ182 variant) is one that anneals atnucleotides 2358 to 2339 of SEQ ID NO:4 (i.e., the site corresponding tothe 182 nucleotide insertion in SEQ ID NO:1). The absolute abundance ofthe Δ182 hTRT mRNA species or its relative abundance compared to thespecies encoding the full-length hTRT protein can be analyzed forcorrelation to cell state (e.g., capacity for indefinite proliferation).It will be appreciated that numerous other primers or amplification ordetection methods can be selected based on the present disclosure. TABLE4 ILLUSTRATIVE PRIMERS Δ182 species (e.g., SEQ ID NO:4) specific primer:5′-GGCACTGGACGTAGGACGTG-3 (SEQ ID NO:550) hTRT (SEQ ID NO:1) specificprimer (TCP1.73): 5′-CACTGCTGGCCTCATTCAGGG-3 (SEQ ID NO:445) Common(forward) primer (TCP1.25): 5′-TACTGCGTGCGTCGGTATG-3′ (SEQ ID NO:399)

[0485] Other variant hTRT genes or gene products that can be detectedinclude those characterized by premature stop codons, deletions,substitutions or insertions. Deletions can be detected by the decreasedsize of the gene, mRNA transcript, or cDNA. Similarly, insertions can bedetected by the increased size of the gene, mRNA transcript, or cDNAinsertions and deletions could also cause shifts in the reading framethat lead to premature stop codons or longer open reading frames.Substitutions, deletions, and insertions can also be detected by probehybridization. Alterations can also be detected by observing changes inthe size of the variant hTRT polypeptide (e.g., by Western analysis) orby hybridization or specific amplification as appropriate.Alternatively, mutations can be determined by sequencing of the gene orgene product according to standard methods. In addition, and as notedabove, amplification assays and hybridization probes can be selected totarget particular abnormalities specifically. For example, nucleic acidprobes or amplification primers can be selected that specificallyhybridize to or amplify, respectively, the region encompassing thedeletion, substitution, or insertion. Where the hTRT gene harbors such amutation, the probe will either (1) fail to hybridize or theamplification reaction will fail to provide specific amplification orcause a change in the size of the amplification product or hybridizationsignal; or (2) the probe or amplification reaction encompasses theentire deletion or either end of the deletion (deletion junction); or(3) similarly, probes and amplification primers can be selected thatspecifically target point mutations or insertions.

[0486] 5) Detection of Mutant hTRT Alleles

[0487] Mutations in the hTRT gene can be responsible for diseaseinitiation or can contribute to a disease condition. Alterations of thegenomic DNA of hTRT can affect levels of gene transcription, changeamino acid residues in the hTRT protein, cause truncated hTRTpolypeptides to be produced, alter pre-mRNA processing pathways (whichcan alter hTRT mRNA levels), and cause other consequences as well.

[0488] Alterations of genomic DNA in non-hTRT loci can also affectexpression of hTRT or telomerase by altering the enzymes or cellularprocesses that are responsible for regulating hTRT, hTR, andtelomerase-associated protein expression and processing and RNP assemblyand transport. Alterations which affect hTRT expression, processing, orRNP assembly could be important for cancer progression, for diseases ofaging, for DNA damage diseases, and others.

[0489] Detection of mutations in hTRT mRNA or its gene and gene controlelements can be accomplished in accordance with the methods herein inmultiple ways. Illustrative examples include the following: A techniquetermed primer screening can be employed; PCR primers are designed whose3′termini anneal to nucleotides in a sample DNA (or RNA) that arepossibly mutated. If the DNA (or RNA) is amplified by the primers, thenthe 3′termini matched the nucleotides in the gene; if the DNA is notamplified, then one or both termini did not match the nucleotides in thegene, indicating a mutation was present. Similar primer design can beused to assay for point mutations using the Ligase Chain Reaction (LCR,described supra). Restriction fragment length polymorphism, RFLP(Pourzand, C., Cerutti, P. (1993) Mutat. Res 288: 113-121), is anothertechnique that can be applied in the present method. A Southern blot ofhuman genomic DNA digested with various restriction enzymes is probedwith an hTRT specific probe. Differences in the fragment number or sizesbetween the sample and a control indicate an alteration of theexperimental sample, usually an insertion or deletion. Single strandconformation polymorphism, SSCP (Orrita, M., et al. (1989) PNAS USA86:2766-70), is another technique that can be applied in the presentmethod. SSCP is based on the differential migration of denaturedwild-type and mutant single-stranded DNA (usually generated by PCR).Single-stranded DNA will take on a three-dimensional conformation thatis sequence-specific. Sequence differences as small as a single basechange can result in a mobility shift on a nondenaturing gel. SSCP isone of the most widely used mutation screening methods because of itssimplicity. Denaturing Gradient Gel Electrophoresis, DGGE (Myers, R. M.,Maniatis, T. and Lerman, L., (1987) Methods in Enzymology, 155:501-527), is another technique that can be applied in the presentmethod. DGGE identifies mutations based on the melting behavior ofdouble-stranded DNA. Specialized denaturing electrophoresis equipment isutilized to observe the melting profile of experimental and controlDNAs: a DNA containing a mutation will have a different mobilitycompared to the control in these gel systems. The examples discussedillustrate commonly employed methodology; many other techniques existwhich are known by those skilled in the art and can be applied inaccordance with the teachings herein.

[0490] F. Karyotype Analysis

[0491] The present invention further provides methods and reagents forkaryotype or other chromosomal analysis using hTRT-sequence probesand/or detecting or locating hTRT gene sequences in chromosomes from ahuman patient, human cell line, or non-human cell. In one embodiment,amplification (i.e., change in copy number), deletion (i.e., partialdeletion), insertion, substitution, or changes in the chromosomallocation (e.g., translocation) of an hTRT gene may be correlated withthe presence of a pathological condition or a predisposition todeveloping a pathological condition (e.g., cancer).

[0492] It has been determined by the present inventors that, in normalhuman cells, the hTRT gene maps close to the telomere of chromosome 5p(see Example 5, infra). The closest STS marker is D5S678 (see FIG. 8).The location can be used to identify markers that are closely linked tothe hTRT gene. The markers can be used to identify YACs, STSs, cosmids,BACs, lambda or P1 phage, or other clones which contain hTRT genomicsequences or control elements. The markers or the gene location can beused to scan human tissue samples for alterations in the normal hTRTgene location, organization or sequence that is associated with theoccurrence of a type of cancer or disease. This information can be usedin a diagnostic or prognostic manner for the disease or cancer involved.Moreover, the nature of any alterations to the hTRT gene can beinformative as to the nature by which cells become immortal. Forinstance, a translocation event could indicate that activation of hTRTexpression occurs in some cases by replacing the hTRT promoter withanother promoter which directs hTRT transcription in an inappropriatemanner. Methods and reagents of the invention of this type can be usedto inhibit hTRT activation. The location may also be useful fordetermining the nature of hTRT gene repression in normal somatic cells,for instance, whether the location is part of non-expressingheterochromatin. Nuclease hypersensitivity assays for distinguishingheterochromatin and euchromatin are described, for example, in Wu etal., 1979, Cell 16:797; Groudine and Weintraub, 1982, Cell 30:131 Grossand Garrard, 1988, Ann. Rev. Biochem. 57:159.

[0493] In one embodiment, alterations to the hTRT gene are identified bykaryotype analysis, using any of a variety of methods known in the art.One useful technique is in situ hybridization (ISH). Typically, when insitu hybridization techniques are used for karyotype analysis, adetectable or detectably-labeled probe is hybridized to a chromosomalsample in situ to locate an hTRT gene sequence. Generally, ISH comprisesone or more of the following steps: (1) fixation of the tissue, cell orother biological structure to be analyzed; (2) prehybridizationtreatment of the biological structure to increase accessibility oftarget DNA (e.g., denaturation with heat or alkali), and to reducenonspecific binding (e.g., by blocking the hybridization capacity ofrepetitive sequences, e.g., using human genomic DNA); (3) hybridizationof one or more nucleic acid probes (e.g., conventional nucleic acids,PNAs, or probes containing other nucleic acid analogs) to the nucleicacid in the biological structure or tissue; (4) posthybridization washesto remove nucleic acid fragments not bound in the hybridization; and,(5) detection of the hybridized nucleic acid fragments. The reagentsused in each of these steps and conditions for their use vary dependingon the particular application. It will be appreciated that these stepscan be modified in a variety of ways well known to those of skill in theart.

[0494] In one embodiment of ISH, the hTRT probe is labeled with afluorescent label (fluorescent in situ hybridization; “FISH”).Typically, it is desirable to use dual color fluorescent in situhybridization, in which two probes are utilized, each labeled by adifferent fluorescent dye. A test probe that hybridizes to the hTRTsequence of interest is labeled with one dye, and a control probe thathybridizes to a different region is labeled with a second dye. A nucleicacid that hybridizes to a stable portion of the chromosome of interest,such as the centromere region, can be used as the control probe. In thisway, one can account for differences between efficiency of hybridizationfrom sample to sample.

[0495] The ISH methods for detecting chromosomal abnormalities (e.g.,FISH) can be performed on nanogram quantities of the subject nucleicacids. Paraffin embedded normal tissue or tumor sections can be used, ascan fresh or frozen material, tissues, or sections. Because FISH can beapplied to limited material, touch preparations prepared from unculturedprimary tumors can also be used (see, e.g., Kallioniemi et al., 1992,Cytogenet. Cell Genet. 60:190). For instance, small biopsy tissuesamples from tumors can be used for touch preparations (see, e.g.,Kallioniemi et al., supra). Small numbers of cells obtained fromaspiration biopsy or cells in bodily fluids (e.g., blood, urine, sputumand the like) can also be analyzed. For prenatal diagnosis, appropriatesamples will include amniotic fluid, maternal blood, and the like.Useful hybridization protocols applicable to the methods and reagentsdisclosed here are described in Pinkel et al., 1988, Proc. Natl. Acad.Sci. USA, 85:9138; EPO Pub. No. 430,402; Choo, ed., METHODS IN MOLECULARBIOLOGY VOL. 33: IN SITU HYBRIDIZATION PROTOCOLS, Humana Press, Totowa,N.J., (1994); and Kallioniemi et al., supra.

[0496] Other techniques useful for karyotype analysis include, forexample, techniques such as quantitative Southern blotting, quantitativePCR, or comparative genomic hybridization (Kallioniemi et al., 1992,Science, 258:818), using the hTRT probes and primers of the inventionwhich may be used to identify amplification, deletion, insertion,substitution or other rearrangement of hTRT sequences in chromosomes ina biological sample.

[0497] G. TRT Polypeptide Assays

[0498] 1) Generally

[0499] The present invention provides methods and reagents for detectingand quantitating hTRT polypeptides. These methods include analyticalbiochemical methods such as electrophoresis, mass spectroscopy, gelshift, capillary electrophoresis, chromatographic methods such as sizeexclusion chromatography, high performance liquid chromatography (HPLC),thin layer chromatography (TLC), hyperdiffusion chromatography, and thelike, or various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting, mass spectrometry, andothers described below and apparent to those of skill in the art uponreview of this disclosure.

[0500] 2) Electrophoretic Assays

[0501] In one embodiment, the hTRT polypeptides are detected in anelectrophoretic protein separation; in one aspect, a two-dimensionalelectrophoresis system is employed. Means of detecting proteins usingelectrophoretic techniques are well known to those of skill in the art(see generally, R. Scopes (1982) PROTEIN PURIFICATION, Springer-Verlag,N.Y.; Deutscher, (1990) METHODS IN ENZYMOLOGY VOL. 182: GUIDE TO PROTEINPURIFICATION, Academic Press, Inc., N.Y.).

[0502] In a related embodiment, a mobility shift assay (see, e.g.,Ausubel et al., supra) is used. For example, labeled-hTR will associatewith hTRT and migrate with altered mobility upon electrophoresis in anondenaturing polyacrylamide gel or the like. Thus, for example, if an(optionally labeled) hTR probe or a (optionally labeled) telomeraseprimer is mixed with a sample containing hTRT, or coexpressed with hTRT(e.g., in a cell-free expression system) the presence of hTRT protein(or a polynucleotide encoding hTRT) in the sample will result in adetectable alteration of hTR mobility.

[0503] 3) Immunoassays

[0504] a) Generally

[0505] The present invention also provides methods for detection of hTRTpolypeptides employing one or more antibody reagents of the invention(i.e., immunoassays). As used herein, an immunoassay is an assay thatutilizes an antibody (as broadly defined herein and specificallyincludes fragments, chimeras and other binding agents) that specificallybinds an hTRT polypeptide or epitope. Antibodies of the invention may bemade by a variety of means well known to those of skill in the art,e.g., as described supra.

[0506] A number of well established immunological binding assay formatssuitable for the practice of the invention are known (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g.,METHODS IN CELL BIOLOGY VOLUME 37: ANTIBODIES IN CELL BIOLOGY, Asai, ed.Academic Press, Inc. New York (1993); BASIC AND CLINICAL IMMUNOLOGY 7thEdition, Stites & Terr, eds. (1991); Harlow and Lane, supra [e.g.,Chapter 14], and Ausubel et al., supra, [e.g., Chapter 11], each ofwhich is incorporated by reference in its entirety and for all purposes.Typically, immunological binding assays (or immunoassays) utilize a“capture agent” to specifically bind to and, often, immobilize theanalyte. In one embodiment, the capture agent is a moiety thatspecifically binds to an hTRT polypeptide or subsequence, such as ananti-hTRT antibody. In an alternative embodiment, the capture agent maybind an hTRT-associated protein or RNA under conditions in which thehTRT-associated molecule remains bound to the hTRT (such that if thehTRT-associated molecule is immobilized the hTRT protein is similarlyimmobilized). It will be understood that in assays in which anhTRT-associated molecule is captured the associated hTRT protein willusually be present and so can be detected, e.g., using an anti-hTRTantibody or the like. Immunoassays for detecting protein complexes areknown in the art (see, e.g., Harlow and Lane, supra, at page 583).

[0507] Usually the hTRT gene product being assayed is detected directlyor indirectly using a detectable label. The particular label ordetectable group used in the assay is usually not a critical aspect ofthe invention, so long as it does not significantly interfere with thespecific binding of the antibody or antibodies used in the assay. Thelabel may be covalently attached to the capture agent (e.g., an anti-TRTantibody), or may be attached to a third moiety, such as anotherantibody, that specifically binds to, e.g.,: the hTRT polypeptide (at adifferent epitope than recognized by the capture agent), the captureagent (e.g., an anti-(first antibody) immunoglobulin); an anti-TRTantibody; an antibody that binds an anti-TRT antibody; or, anantibody/telomerase complex (e.g., via binding to an associated moleculesuch as a telomerase-associated protein). Other proteins capable ofbinding an antibody used in the assay, such as protein A or protein G,may also be labeled. In some embodiments, it will be useful to use morethan one labeled molecule (i.e., ones that can be distinguished from oneanother). In addition, when the target bound (e.g., immobilized) by thecapture agent (e.g., anti-hTRT antibody) is a complex (i.e., a complexof hTRT and a TRT-associated protein, hTR, or other TRT associatedmolecule), a labeled antibody that recognizes the protein or RNAassociated with the hTRT protein can be used. When the complex is aprotein-nucleic acid complex (e.g., TRT-hTR), the reporter molecule canbe a polynucleotide or other molecule (e.g., enzyme) that recognizes theRNA component of the complex.

[0508] Some immunoassay formats do not require the use of labeledcomponents. For instance, agglutination assays can be used to detect thepresence of the target antibodies. In this case, antigen-coatedparticles are agglutinated by samples comprising the target antibodies.In this format, the components do not need to be labeled, and thepresence of the target antibody can be detected by simple visualinspection.

[0509] b) Non-Competitive Assay Formats

[0510] The present invention provides methods and reagents forcompetitive and noncompetitive immunoassays for detecting hTRTpolypeptides. Noncompetitive immunoassays are assays in which the amountof captured analyte (in this case hTRT) is directly measured. One suchassay is a two-site, monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two non-interfering epitopes on the hTRT protein.See, e.g., Maddox et al., 1983, J. Exp. Med., 158:1211 for backgroundinformation. In one preferred “sandwich” assay, the capture agent (e.g.,an anti-TRT antibody) is bound directly to a solid substrate where it isimmobilized. These immobilized antibodies then capture any hTRT proteinpresent in the test sample. The hTRT thus immobilized can then belabeled, i.e., by binding to a second anti-hTRT antibody bearing alabel. Alternatively, the second anti-hTRT antibody may lack a label,but be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second antibodyalternatively can be modified with a detectable moiety, such as biotin,to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

[0511] c) Competitive Assay Formats

[0512] In competitive assays, the amount of hTRT protein present in thesample is measured indirectly by measuring the amount of an added(exogenous) hTRT displaced (or competed away) from a capture agent(e.g., anti-TRT antibody) by the hTRT protein present in the sample. Inone competitive assay, a known amount of labeled hTRT protein is addedto the sample and the sample is then contacted with a capture agent(e.g., an antibody that specifically binds hTRT protein). The amount ofexogenous (labeled) hTRT protein bound to the antibody is inverselyproportional to the concentration of hTRT protein present in the sample.In one embodiment, the antibody is immobilized on a solid substrate. Theamount of hTRT protein bound to the antibody may be determined either bymeasuring the amount of hTRT protein present in a TRT/antibody complex,or alternatively by measuring the amount of remaining uncomplexed TRTprotein. The amount of hTRT protein may be detected by providing alabeled hTRT molecule.

[0513] A hapten inhibition assay is another example of a competitiveassay. In this assay hTRT protein is immobilized on a solid substrate. Aknown amount of anti-TRT antibody is added to the sample, and the sampleis then contacted with the immobilized hTRT protein. In this case, theamount of anti-TRT antibody bound to the immobilized hTRT protein isinversely proportional to the amount of hTRT protein present in thesample. The amount of immobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. In this aspect, detection may bedirect, where the antibody is labeled, or indirect where the label isbound to a molecule that specifically binds to the antibody as describedabove.

[0514] d) Other Assay Formats

[0515] The invention also provides reagents and methods for detectingand quantifying the presence of hTRT in the sample by using animmunoblot (Western blot) format. In this format, hTRT polypeptides in asample are separated from other sample components by gel electrophoresis(e.g., on the basis of molecular weight), the separated proteins aretransferred to a suitable solid support (such as a nitrocellulosefilter, a nylon filter, derivatized nylon filter, or the like), and thesupport is incubated with anti-TRT antibodies of the invention. Theanti-TRT antibodies specifically bind to hTRT or other TRT on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) or other labeling reagents that specifically bindto the anti-TRT antibody.

[0516] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals canthen be detected according to standard techniques (see, Monroe et al.,1986, Amer. Clin. Prod. Rev. 5:34).

[0517] As noted supra, assay formats using FACS (and equivalentinstruments or methods) have advantages when measuring hTRT geneproducts in a heterogeneous sample (such as a biopsy sample containingboth normal and malignant cells).

[0518] e) Substrates, Solid Supports, Membranes, Filters

[0519] As noted supra, depending upon the assay, various components,including the antigen, target antibody, or anti-hTRT antibody, may bebound to a solid surface or support (i.e., a substrate, membrane, orfilter paper). Many methods for immobilizing biomolecules to a varietyof solid surfaces are known in the art. For instance, the solid surfacemay be a membrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC,polypropylene, or polystyrene), a test tube (glass or plastic), adipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, and thelike), a microcentrifuge tube, or a glass or plastic bead. The desiredcomponent may be covalently bound or noncovalently attached throughnonspecific bonding.

[0520] A wide variety of organic and inorganic polymers, both naturaland synthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

[0521] In preparing the surface, a plurality of different materials maybe employed, particularly as laminates, to obtain various properties.For example, protein coatings, such as gelatin can be used to avoidnon-specific binding, simplify covalent conjugation, enhance signaldetection or the like.

[0522] If covalent bonding between a compound and the surface isdesired, the surface will usually be polyfunctional or be capable ofbeing polyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas (1970) J. Biol. Chem. 2453059).

[0523] In addition to covalent bonding, various methods fornoncovalently binding an assay component can be used. Noncovalentbinding is typically nonspecific absorption of a compound to thesurface.

[0524] One of skill in the art will appreciate that it is oftendesirable to reduce non-specific binding in immunoassays. Particularly,where the assay involves an antigen or antibody immobilized on a solidsubstrate it is desirable to minimize the amount of non-specific bindingto the substrate. Means of reducing such non-specific binding are wellknown to those of skill in the art. Typically, this involves coating thesubstrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk sometimes preferred.Alternatively, the surface is designed such that it nonspecificallybinds one component but does not significantly bind another. Forexample, a surface bearing a lectin such as Concanavalin A will bind acarbohydrate containing compound but not a labeled protein that lacksglycosylation. Various solid surfaces for use in noncovalent attachmentof assay components are reviewed in U.S. Pat. Nos. 4,447,576 and4,254,082.

[0525] H) Assays for Anti-TRT Antibodies

[0526] The present invention also provides reagents and assays fordetecting hTRT-specific immunoglobulins. In one embodiment, immobilizedhTRT (e.g., recombinant hTRT bound to a microassay plate well) isincubated with serum from a patient under conditions in which anti-hTRTantibodies, if present, bind the immobilized hTRT. After washing toremove nonspecifically bound immunoglobulin, bound serum antibodies canbe detected, if they are present, by adding detectably labeledanti-(human Ig) antibodies (alternative embodiments and variations arewell known to those of skill in the art; see, e.g., Harlow, supra, atCh. 14). These assays are useful for detecting anti-hTRT antibodies inany source including animal or human serum or a carrier such as saline.In one embodiment, the assays are used to detect or monitor an immuneresponse to hTRT proteins in a patient, particularly an autoimmune(e.g., anti-telomerase) response. Anti-hTRT antibodies may be present inthe serum or other tissues or fluids from a patient suffering from anautoimmune disease or other condition.

[0527] I) Assay Combinations

[0528] The diagnostic and prognostic assays described herein can becarried out in various combinations and can also be carried out inconjunction with other diagnostic or prognostic tests. For example, whenthe present methods are used to detect the presence of cancer cells inpatient sample, the presence of hTRT can be used to determine the stageof the disease, whether a particular tumor is likely to invade adjoiningtissue or metastasize to a distant location, and whether a recurrence ofthe cancer is likely. Tests that may provide additional informationinclude microscopic analysis of biopsy samples, detection of antigens(e.g., cell-surface markers) associated with tumorigenicity (e.g., usinghistocytochemistry, FACS, or the like), imaging methods (e.g., uponadministration to a patient of labeled anti-tumor antibodies),telomerase activity assays, telomere length assays, hTR assays, or thelike. Such combination tests can provide useful information regardingthe progression of a disease.

[0529] It will also be recognized that combinations of assays canprovide useful information. For example, and as noted above, assays forhTRT mRNA can be combined with assays for hTR (human telomerase RNA) ortelomerase activity (i.e., TRAP) assays to provide information abouttelomerase assembly and function.

[0530] J) Kits

[0531] The present invention also provides kits useful for thescreening, monitoring, diagnosis and prognosis of patients with atelomerase-related condition, or for determination of the level ofexpression of hTRT in cells or cell lines. The kits include one or morereagents for determining the presence or absence of an hTRT gene product(RNA or protein) or for quantifying expression of the hTRT gene.Preferred reagents include nucleic acid primers and probes thatspecifically bind to the hTRT gene, RNA, cDNA, or portions thereof,along with proteins, peptides, antibodies, and control primers, probes,oligonucleotides, proteins, peptides and antibodies. Other materials,including enzymes (e.g., reverse transcriptases, DNA polymerases,ligases), buffers, reagents (labels, dNTPs), may be included.

[0532] The kits may include alternatively, or in combination with any ofthe other components described herein, an antibody that specificallybinds to hTRT polypeptides or subsequences thereof. The antibody can bemonoclonal or polyclonal. The antibody can be conjugated to anothermoiety such as a label and/or it can be immobilized on a solid support(substrate). The kit(s) may also contain a second antibody for detectionof hTRT polypeptide/antibody complexes or for detection of hybridizednucleic acid probes, as well as one or more hTRT peptides or proteinsfor use as control or other reagents.

[0533] The antibody or hybridization probe may be free or immobilized ona solid support such as a test tube, a microtiter plate, a dipstick andthe like. The kit may also contain instructional materials teaching theuse of the antibody or hybridization probe in an assay for the detectionof TRT. The kit may contain appropriate reagents for detection oflabels, or for labeling positive and negative controls, washingsolutions, dilution buffers and the like.

[0534] In one embodiment, the kit includes a primer pair for amplifyinghTRT mRNA. Such a kit may also include a probe for hTRT amplified DNAand/or a polymerase, buffer, dNTPs, and the like. In another, the kitcomprises a probe, optionally a labeled probe. In another, the kitcomprises an antibody.

[0535] X. Identification of Modulators of Telomerase Activity

[0536] A. Generally

[0537] The invention provides compounds and treatments that modulate theactivity or expression of a telomerase or telomerase component (e.g.,hTRT protein). The invention also provides assays and screening methods(including high-throughput screens) for identification of compounds andtreatments that modulate telomerase activity or expression. Thesemodulators of telomerase activity and expression (hereinafter referredto as “modulators”) include telomerase agonists (which increasetelomerase activity and/or expression) and telomerase antagonists (whichdecrease telomerase activity and/or expression).

[0538] The modulators of the invention have a wide variety of uses. Forexample, it is contemplated that telomerase modulators will be effectivetherapeutic agents for treatment of human diseases. Screening foragonist activity and transcriptional or translational activatorsprovides for compositions that increase telomerase activity in a cell(including a telomere dependent replicative capacity, or a “partial”telomerase activity). Such agonist compositions provide for methods ofimmortalizing otherwise normal untransformed cells, including cellswhich can express useful proteins. Such agonists can also provide formethods of controlling cellular senescence. Conversely, screening forantagonist activity proyides for compositions that decrease telomeredependent replicative capacity, thereby mortalizing otherwise immortalcells, such as cancer cells. Screening for antagonist activity providesfor compositions that decrease telomerase activity, thereby preventingunlimited cell division of cells exhibiting unregulated cell growth,such as cancer cells. Illustrative diseases and conditions that may betreated using modulators are listed herein, e.g., in Sections VIII andIX, supra. In general, the modulators of the invention can be usedwhenever it is desired to increase or decrease a telomerase activity ina cell or organism. Thus, in addition to use in treatment of disease, amodulator that increases hTRT expression levels can be used to produce acultured human cell line having properties as generally described inSection VIII, supra, and various other uses that will be apparent to oneof skill.

[0539] A compound or treatment modulates “expression” of telomerase or atelomerase component when administration of the compound or treatmentchanges the rate or level of transcription of the gene encoding atelomerase component (e.g., the gene encoding hTRT mRNA), affectsstability or post-transcriptional processing of RNA encoding atelomerase component (e.g., transport, splicing, polyadenylation, orother modification), affects translation, stability, post-translationalprocessing or modification of an encoded protein (e.g., hTRT), orotherwise changes the level of functional (e.g., catalytically active)telomerase RNP. A compound or treatment affects a telomerase “activity”when administration of the compound or treatment changes a telomeraseactivity such as any activity described in Section IV(B), supra (e.g.,including processive or non-processive telomerase catalytic activity;telomerase processivity; conventional reverse transcriptase activity;nucleolytic activity; primer or substrate binding activity; dNTP bindingactivity; RNA binding activity; telomerase RNP assembly; and proteinbinding activity). It will be appreciated that there is not necessarilya sharp delineation between changes in “activity” and changes in“expression,” and that these terms are used for ease of discussion andnot for limitation. It will also be appreciated that the modulators ofthe invention should specifically affect telomerase activity orexpression (e.g., without generally changing the expression ofhousekeeping proteins such as actin) rather than, for example, reducingexpression of a telomerase component by nonspecific poisoning of atarget cell.

[0540] B. Assays for Identification of Telomerase Modulators

[0541] The invention provides methods and reagents to screen forcompositions or compounds capable of affecting expression of atelomerase or telomerase component, capable of modifying the DNAreplicative capacity of telomerase, or otherwise modifying the abilityof the telomerase enzyme and TRT protein to synthesize telomeric DNA(“full activity”). The invention also provides screens for modulators ofany or all of hTRT's “partial activities.” Thus, the present inventionprovides assays that can be used to screen for agents that increase theactivity of telomerase, for example, by causing hTRT protein ortelomerase to be expressed in a cell in which it normally is notexpressed or by increasing telomerase activity levels in telomerasepositive cells.

[0542] Telomerase or telomerase subunit proteins or their catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening therapeutic compounds in any of a variety of drug screeningtechniques. The fragment employed in such a test may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweentelomerase or the subunit protein and the agent being tested, may bemeasured.

[0543] In various embodiments, the invention includes methods forscreening for antagonists that: bind to the enzyme's active site;inhibit the association of its RNA moiety, telomerase-associatedproteins, nucleotides, or telomeric DNA to telomerase or hTRT protein;promote the disassociation of the enzyme complex; interfere withtranscription of the telomerase RNA moiety (e.g., hTR); or inhibit anyof the “partial activities” described herein. The invention providesmethods for screening for compositions that inhibit the association ofnucleic acid and/or telomerase-associated compositions with hTRT, suchas the association of hTR with hTRT or the association of hTRT with thehuman homologs of p80 or p95 or another associated protein, orassociation of hTRT with a telomere or a nucleotide; screening forcompositions that promote the disassociation or promote the association(i.e., assembly) of the enzyme complex, such as an antibody directed tohTR or hTRT; screening for agents that effect the processivity of theenzyme; and screening for nucleic acids and other compositions that bindto telomerase, such as a nucleic acid complementary to hTR. Theinvention further contemplates screening for compositions that increaseor decrease the transcription of the hTRT gene and/or translation of thehTRT gene product. The invention also contemplates a method of screeningfor telomerase modulators in animals, in one embodiment, byreconstituting a telomerase activity, or an anti-telomerase activity, inan animal, such as a transgenic animal. The invention provides for invivo assays systems that include “knockout” models, in which one orseveral units of the endogenous telomerase, telomerase RNA moiety and/ortelomerase-associated proteins have been deleted or inhibited. Theendogenous telomerase activity, full or partial, can remain or beabsent. In one embodiment, an exogenous telomerase activity, full orpartial, is reconstituted.

[0544] In one embodiment of the invention, a variety of partial activitytelomerase assays are provided to identify a variety of differentclasses of modulators of telomerase activity. The “partial activity”assays of the invention allow identification of classes of telomeraseactivity modulators that might otherwise not be detected in a “fullactivity” telomerase assay. One partial activity assay involves thenon-processive activity of TRT and telomerase. The processive nature oftelomerase is described by Morin (1989) Cell 59:521-529; see also Prowse(1993) “Identification of a nonprocessive telomerase activity from mousecells” Proc. Natl. Acad. Sci. USA 90:1493-1497. Another partial activityassay of the invention exploits the “reverse-transcriptase-like”activity of telomerase. In these assays, one assays the reversetranscriptase activity of the hTRT protein. See Lingner (1997) “Reversetranscriptase motifs in the catalytic subunit of telomerase” Science276:561-567. Another partial activity assay of the invention exploitsthe “nucleolytic activity” of hTRT and telomerase, involving theenzyme's removing of at least one nucleotide, typically guanosine, fromthe 3′ strand of a primer. This nucleolytic activity has been observedin Tetrahymena telomerase by Collins (1993) “Tetrahymena telomerasecatalyzes nucleolytic cleavage and nonprocessive elongation” Genes Dev7:1364-1376. Another partial activity assay of the invention involvesanalyzing hTRT's and telomerase's ability to bind nucleotides as part ofits enzymatic processive DNA polymerization activity. Another partialactivity assay of the invention involves analyzing hTRT's ortelomerase's ability to bind its RNA moiety, i.e., hTR for human cells,used as a template for telomere synthesis. Additional partial activityassays of the invention involve analyzing hTRT's and telomerase'sability to bind chromosomes in vivo, or to bind oligonucleotide primersin vitro or in reconstituted systems, or to bind proteins associatedwith chromosomal structure (see, for an example of such a protein,Harrington (1995) J Biol Chem 270: 8893-8901). Chromosomal structureswhich bind hTRT include, for example, telomeric repeat DNA, telomereproteins, histones, nuclear matrix protein, cell division/cell cyclecontrol proteins and the like.

[0545] In one embodiment, an assay for identification of modulatorscomprises contacting one or more cells (i.e., “test cells”) with a testcompound, and determining whether the test compound affects expressionor activity of a telomerase (or telomerase component) in the cell.Usually this determination comprises comparing the activity orexpression in the test cell compared to a similar cell or cells (i.e.,control cells) that have not been contacted with the test compound.Alternatively, cell extracts may be used in place of intact cells. In arelated embodiment, the test compound is administered to a multicellularorganism (e.g., a plant or animal). The telomerase or telomerasecomponent may be wholly endogenous to the cell or multicellular organism(i.e., encoded by naturally occurring endogenous genes), or may be arecombinant cell or transgenic organism comprising one or morerecombinantly expressed telomerase components (e.g., hTRT, hTR,telomerase-associated proteins), or may have both endogenous andrecombinant components. Thus, in one embodiment,telomerase-activity-modulators are administered to mortal cells. Inanother embodiment, telomerase-activity-modulators are administered toimmortal cells. For example, antagonists of telomerase-mediated DNAreplication can be identified by administering the putative inhibitorycomposition to a cell that is known to exhibit significant amounts oftelomerase activity, such as cancer cells, and measuring whether adecrease in telomerase activity, telomere length, or proliferativecapacity is observed, all of which are indicative of a compound withantagonist activity.

[0546] In another embodiment, a modulator is identified by monitoring achange in a telomerase activity of a ribonucleoprotein complex (RNP)comprising a TRT (e.g., hTRT) and a template RNA (e.g., hTR), which RNPis reconstituted in vitro (e.g., as described in Example 7, infra).

[0547] In yet another embodiment, the modulator is identified bymonitoring a change in expression of a TRT gene product (e.g., RNA orprotein) in a cell, animal, in vitro expression system, or otherexpression system.

[0548] In still another embodiment, the modulator is identified bychanging the expression of a reporter gene, such as that described inExample 15, whose expression is regulated, in whole or part, by anaturally occurring TRT regulatory element such as a promoter orenhancer. In a related embodiment, the ability of a test compound tobind to a telomerase component (e.g., hTRT), RNA, or gene regulatorysequence (e.g., the TRT gene promoter) is assayed.

[0549] In another embodiment, the modulator is identified by observingchanges in hTRT pre-mRNA processing, for example, alternatively splicedproducts, alternative poly-adenylation events, RNA cleavage, and thelike. In a related embodiment the activity of the modulator can beobserved by monitoring the production of variant hTRT polypeptides, someof which may possess dominant-negative telomerase regulation activity.

[0550] Assay formats for identification of compounds that affectexpression and activity of proteins are well known in thebiotechnological and pharmaceutical industries, and numerous additionalassays and variations of the illustrative assays provided supra will beapparent to those of skill.

[0551] Changes in telomerase activity or expression can be measured byany suitable method. Changes in levels of expression of a telomerasecomponent (e.g., hTRT protein) or precursor (e.g., hTRT mRNA) can beassayed using methods well known to those of skill, some of which aredescribed hereinabove, e.g., in Section 1× and including monitoringlevels of TRT gene products (e.g., protein and RNAs) by hybridization(e.g., using the TRT probes and primers of the invention), immunoassays(e.g., using the anti-TRT antibodies of the invention), RNAse protectionassays, amplification assays, or any other suitable detection meansdescribed herein or known in the art. Quantitating amounts of nucleicacid in a sample (e.g., evaluating levels of RNA, e.g., hTR or hTRTmRNA) is also useful in evaluating cis- or trans-transcriptionalregulators.

[0552] Similarly, changes in telomerase activity can be measured usingmethods such as those described herein (e.g., in Section IV(B), supra)or other assays of telomerase function. Quantitation of telomeraseactivity, when desired, may be carried out by any method, includingthose disclosed herein. Telomerase antagonists that can cause oraccelerate loss of telomeric structure can be identified by monitoringand measuring their effect on telomerase activity in vivo, ex vivo, orin vitro, or by their effects on telomere length (as measured ordetected through staining, use of tagged hybridization probes or othermeans) or, simply, by the inhibition of cell division of telomerasepositive cancer cells (critical shortening of telomeres leads to aphenomenon termed “crisis” or M2 senescence (Shay, 1991) Biochem.Biophys. Acta 1072:1-7), which cancer cells have bypassed by theactivation of telomerase, but which, in the absence of telomerase, willlead to their senescence or death through chromosomal deletion andrearrangement). The in vivo human telomerase activity reconstitutionprovides for a method of screening for telomerase modulators in cells oranimals from any origin. Such agonists can be identified in an activityassay of the invention, including measurements of changes in telomerelength. Other examples of assays measuring telomerase activity in cellsinclude assays for the accumulation or loss of telomere structure, theTRAP assay or a quantitative polymerase chain reaction assay.

[0553] In one embodiment, the assays of the invention also include amethod where the test compound produces a statistically significantdecrease in the activity of hTRT as measured by the incorporation of alabeled nucleotide into a substrate compared to the relative amount ofincorporated label in a parallel reaction lacking the test compound,thereby determining that the test compound is a telomerase inhibitor.

[0554] The methods of the invention are amenable to adaptations fromprotocols described in the scientific and patent literature and known inthe art. For example, when a telomerase or TRT protein of this inventionis used to identify compositions which act as modulators of telomeraseactivities, large numbers of potentially useful molecules can bescreened in a single test. The modulators can have an inhibitory(antagonist) or potentiating (agonist) effect on telomerase activity.For example, if a panel of 1,000 inhibitors is to be screened, all 1,000inhibitors can potentially be placed into one microtiter well and testedsimultaneously. If such an inhibitor is discovered, then the pool of1,000 can be subdivided into 10 pools of 100 and the process repeateduntil an individual inhibitor is identified.

[0555] In drug screening large numbers of compounds are examined fortheir ability to act as telomerase modulators, a process greatlyaccelerated by the techniques of high throughput screening. The assaysfor telomerase activity, full or partial, described herein may beadapted to be used in a high throughput technique. Those skilled in theart appreciate that there are numerous methods for accomplishing thispurpose.

[0556] Another technique for drug screening which may be applied forhigh throughput screening of compounds having suitable binding affinityto the telomerase or telomerase protein subunit is described in detailin “Determination of Amino Acid Sequence Antigenicity” by Geysen,(Geysen, WO Application 84/03564, published on Sep. 13, 1984,incorporated herein by reference). In summary, large numbers ofdifferent small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with fragments of telomerase or telomerase proteinsubunits and washed. Bound telomerase or telomerase protein subunit isthen detected by methods well known in the art. Substantially purifiedtelomerase or telomerase protein subunit can also be coated directlyonto plates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

[0557] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingtelomerase or subunit protein(s) specifically compete with a testcompound for binding telomerase or the subunit protein. Antibodies canalso be used to detect the presence of any peptide which shares one ormore antigenic determinants with the telomerase or subunit protein.

[0558] Additional methods for identifying modulators of a telomeraseactivity have been described in U.S. Pat. No. 5,645,986, which isincorporated herein by reference. It will be appreciated that thepresent invention provides improvements to previously known methods, inpart by providing reagents such as hTRT polynucleotides, probes andprimers, highly purified hTR, hTRT and telomerase, as well asanti-telomerase and anti-TRT antibodies, all of which may be used inassays, e.g., as controls, standards, binding or hybridization agents,or otherwise.

[0559] It will be recognized that the recombinantly produced telomeraseand TRT (e.g., hTRT) of the invention will be useful in assays foridentification of modulators. The screening assay can utilize telomeraseor hTRT derived by a full or partial reconstitution of telomeraseactivity, or by an augmentation of existing activity. The assay orscreens provided by the invention can be used to test for the ability oftelomerase to synthesize telomeric DNA or to test for any one or all orof the “partial activities” of hTRT and TRTs generally, as describedabove. The assay can incorporate ex vivo modification of cells whichhave been manipulated to express telomerase with or without its RNAmoiety or associated proteins, and these can be re-implanted into ananimal, which can be used for in vivo testing. Thus, this inventionprovides in vivo assays and transgenic animals useful therein. These invivo assays systems can employ “knockout” cells, in which one or severalunits of the endogenous telomerase enzyme complex have been deleted orinhibited, as well as cells in which an exogenous or endogenoustelomerase activity is reconstituted or activated.

[0560] Telomerases and TRT proteins that have been modified in asite-specific manner (by site-specific mutation) to modify or delete anyor all functions of the telomerase enzyme or the TRT protein can also beemployed in the screens of the invention to discover therapeutic agents.For example, the TRT can be engineered to lose its ability to bindsubstrate DNA, to bind its RNA moiety (as hTR), to catalyze the additionof telomeric DNA, to bind deoxynucleotide substrate, to have nucleolyticactivity, to bind telomere-associated proteins or chromosomalstructures, and the like. The resulting “mutant proteins” or “muteens”can be used to identify compounds that specifically modulate one,several, or all functions or activities of the TRT protein ortelomerase.

[0561] C. Exemplary Telomerase Modulators

[0562] 1) Generally

[0563] The test compounds referred to supra may be any of a largevariety of compounds, both naturally occurring and synthetic, organicand inorganic, and including polymers (e.g., oligopeptides,polypeptides, oligonucleotides, and polynucleotides), small molecules,antibodies (as broadly defined herein), sugars, fatty acids, nucleotidesand nucleotide analogs, analogs of naturally occurring structures (e.g.,peptide mimetics, nucleic acid analogs, and the like), and numerousother compounds.

[0564] The invention provides modulators of all types, withoutlimitation to any particular mechanism of action. For illustrativepurposes, examples of modulators include compounds or treatments that:

[0565] (i) bind to the hTRT polypeptide (e.g., the active site of theenzyme) or other telomerase component, and affect a telomerase activity;

[0566] (ii) inhibit or promote association, or inhibit or promotedisassociation, of a telomerase component (e.g., hTRT or the hTRT-hTRRNP) with or from a telomerase-associated protein (e.g., including thosedescribed in Section IV(D), supra);

[0567] (iii) inhibit or promote association, or inhibit or promotedisassociation, of telomerase polypeptides (e.g., hTRT) with or from atelomerase RNA (e.g., hTR);

[0568] (iv) inhibit or promote association, or inhibit or promotedisassociation, of telomerase polypeptides (e.g., hTRT) with or fromchromosomes (e.g., telomeres) or chromosomal DNA (e.g. telomeric DNA);

[0569] (v) increase or decrease expression of a telomerase componentgene product (e.g., products of the hTRT gene), including change therate or level of transcription of the TRT gene, or translation,transport or stability of a gene product, or the like, by binding to thegene or gene product (e.g., by interacting with a factor (e.g., atranscription regulatory protein) that affects transcription of the hTRTgene or another telomerase component).

[0570] 2) Peptide Modulators

[0571] Potential modulators of telomerase activity also include peptides(e.g., inhibitory (antagonist) and activator (agonist) peptidemodulators). For example, oligopeptides with randomly generatedsequences can be screened to discover peptide modulators (agonists orinhibitors) of telomerase activity. Such peptides can be used directlyas drugs or to find the orientation or position of a functional groupthat can inhibit telomerase activity that, in turn, leads to design andtesting of a small molecule inhibitor, or becomes the backbone forchemical modifications that increase pharmacological utility. Peptidescan be structural mimetics, and one can use molecular modeling programsto design mimetics based on the characteristic secondary structureand/or tertiary structure of telomerase enzyme and hTRT protein. Suchstructural mimetics can also be used therapeutically, in vivo, asmodulators of telomerase activity (agonists and antagonists). Structuralmimetics can also be used as immunogens to elicit anti-telomerase oranti-TRT protein antibodies.

[0572] 3) Inhibitory Natural Compounds as Modulators of TelomeraseActivity

[0573] In addition, a large number of potentially usefulactivity-modifying compounds can be screened in extracts from naturalproducts as a source material. Sources of such extracts can be from alarge number of species of fungi, actinomyces, algae, insects, protozoa,plants, and bacteria. Those extracts showing inhibitory activity canthen be analyzed to isolate the active molecule. See for example, Turner(1996) J. Ethnopharmacol 51(1-3):39-43; Suh (1995) Anticancer Res.15:233-239.

[0574]4) Inhibitory Oligonucleotides

[0575] One particularly useful set of inhibitors provided by the presentinvention includes oligonucleotides which are able to either bind mRNAencoding hTRT protein or to the hTRT gene, in either case preventing orinhibiting the production of functional hTRT protein. Otheroligonucleotides of the invention interact with telomerase's RNA moiety,such as hTR, or are able to prevent binding of telomerase or hTRT to itsDNA target, or one telomerase component to another, or to a substrate.Such oligonucleotides can also bind the telomerase enzyme, hTRT protein,or both protein and RNA and inhibit a partial activity as describedabove (such as its processive activity, its reverse transcriptaseactivity, its nucleolytic activity, and the like). The association canbe through sequence specific hybridization to another nucleic acid or bygeneral binding, as in an aptamer, or both.

[0576] Telomerase activity can be inhibited by targeting the hTRT mRNAwith antisense oligonucleotides capable of binding the hTRT mRNA.

[0577] Another useful class of inhibitors includes oligonucleotideswhich cause inactivation or cleavage of hTRT mRNA or hTR. That is, theoligonucleotide is chemically modified, or has enzyme activity, whichcauses such cleavage, such as is the case for a ribozyme, anEDTA-tethered oligonucleotide, or a covalently bound oligonucleotide,such as a psoralen or other cross-linking reagent bound oligonucleotide.As noted above, one may screen a pool of many different sucholigonucleotides for those with the desired activity.

[0578] Another useful class of inhibitors includes oligonucleotideswhich bind polypeptides. Double- or single-stranded DNA or double- orsingle-stranded RNA molecules that bind to specific polypeptides targetsare called “aptamers.” The specific oligonucleotide-polypeptideassociation may be mediated by electrostatic interactions. For example,aptamers specifically bind to anion-binding exosites on thrombin, whichphysiologically binds to the polyanionic heparin (Bock (1992) Nature355:564-566). Because hTRT protein binds both hTR and its DNA substrate,and because the present invention provides hTRT and other TRT proteinsin purified form in large quantities, those of skill in the art canreadily screen for TRT-binding aptamers using the methods of theinvention.

[0579] Oligonucleotides (e.g., RNA oligonucleotides) that bindtelomerase, hTRT, hTR, or portions thereof, can be generated using thetechniques of SELEX (Tuerk, 1997, Methods Mol Biol 67, 2190). In thistechnique a very large pool (106-109) of random sequence nucleic acidsis bound to the target (e.g. hTRT) using conditions that cause a largeamount of discrimination between molecules with high affinity and lowaffinity for binding the target. The bound molecules are separated fromunbound, and the bound molecules are amplified by virtue of a specificnucleic acid sequence included at their termini and suitableamplification reagents. This process is reiterated several times until arelatively small number of molecules remain that possess high bindingaffinity for the target. These molecules can then be tested for theirability to modulate telomerase activity as described herein.

[0580] Antagonists of telomerase-mediated DNA replication can also bebased on inhibition of hTR (Norton (1996) Nature Biotechnology14:615-619) through complementary sequence recognition or cleavage, asthrough ribozymes.

[0581] The inhibitory oligonucleotides of the invention can betransferred into the cell using a variety of techniques well known inthe art. For example, oligonucleotides can be delivered into thecytoplasm without specific modification. Alternatively, they can bedelivered by the use of liposomes which fuse with the cellular membraneor are endocytosed, i.e., by employing ligands attached to the liposomeor directly to the oligonucleotide, that bind to surface membraneprotein receptors of the cell resulting in endocytosis. Alternatively,the cells may be permeabilized to enhance transport of theoligonucleotides into the cell, without injuring the host cells. One canuse a DNA binding protein, e.g., HBGF-1, known to transport anoligonucleotide into a cell.

[0582] 5) Inhibitory Ribozymes

[0583] Ribozymes act by binding to a target RNA through the target RNAbinding portion of a ribozyme which is held in close proximity to anenzymatic portion of the ribozyme that cleaves the target RNA. Thus, theribozyme recognizes and binds a target RNA usually through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocleave and inactivate the target RNA. Cleavage of a target RNA in such amanner will destroy its ability to direct synthesis of an encodedprotein if the cleavage occurs in the coding sequence. After a ribozymehas bound and cleaved its RNA target, it is typically released from thatRNA and so can bind and cleave new targets repeatedly.

[0584] 6) Identifying Telomerase-Associated Proteins for Use asModulators

[0585] In one embodiment of the invention, telomerase is used toidentify telomerase-associated proteins, i.e., telomerase accessoryproteins which modulate or otherwise complement telomerase activity. Asnoted above, these proteins or fragments thereof can modulate functionby causing the dissociation or preventing the association of thetelomerase enzyme complex, preventing the assembly of the telomerasecomplex, preventing hTRT from binding to its nucleic acid complement orto its DNA template, preventing hTRT from binding nucleotides, orpreventing, augmenting, or inhibiting any one, several or all of thepartial activities of the telomerase enzyme or hTRT protein, asdescribed above.

[0586] One of skill in the art can use the methods of the invention toidentify which portions (e.g., domains) of these telomerase-associatingproteins contact telomerase. In one embodiment of the invention, thesetelomerase-associating proteins or fragments thereof are used asmodulators of telomerase activity.

[0587] 7) Telomerase-Associated Proteins as Dominant Negative Mutants

[0588] In one embodiment of the invention, telomerase-associatedproteins are used as modulators of telomerase activity.Telomerase-associated proteins include chromosomal structures, such ashistones, nuclear matrix proteins, cell division and cell cycle controlproteins, and the like. Other telomerase-associated proteins which canbe used as modulators for the purpose of the invention include the p80and p95 proteins and their human homologs, such as TP1 and TRF-1 (Chong,1995, Science 270:1663-1667). In addition, fragments of thesetelomerase-associated proteins can be identified by the skilled artisanin accordance with the methods of the invention and used as modulatorsof telomerase activity.

[0589] 8) Dominant Negative Mutants

[0590] Eight highly conserved motifs have been identified between TRTsof different non-human species, as described above (see also Lingner(1997) Science 276:561-567). FIG. 4 shows a schematic of the human TRTamino acid sequence (from pGRN121) and RT motifs as compared to S. pombeTrt1p, Euplotes p123 and S. cerevisiae Est2 p. The present inventionprovides recombinant and synthetic nucleic acids in which the codons forthe conserved amino acid residues in each, alone or in conjunction withone or more additional codons, of all eight of these motifs has been achanged to each of the other codons. A variety of the resulting codingsequences express a non-functional hTRT. See, for instance, Example 16.Thus, the present invention provides, for example, a wide variety of“mutated” telomerase enzymes and TRT proteins which have a partialactivity but not full activity of telomerase. For example, one suchtelomerase is able to bind telomeric structures, but not bindtelomerase-associated RNA (i.e., hTR). If expressed at high enoughlevels, such a telomerase mutant can deplete a necessary telomerasecomponent (e.g., hTR) and thereby function as an inhibitor of wild-typetelomerase activity. A mutated telomerase acting in this manner is anantagonist or a so-called “dominant-negative” mutant.

[0591] 9) Antibodies

[0592] In general, the antibodies of the invention can be used toidentify, purify, or inhibit any or all activity of telomerase enzymeand hTRT protein. Antibodies can act as antagonists of telomeraseactivity in a variety of ways, for example, by preventing the telomerasecomplex or nucleotide from binding to its DNA substrates, by preventingthe components of telomerase from forming an active complex, bymaintaining a functional (telomerase complex) quaternary structure or bybinding to one of the enzyme's active sites or other sites that haveallosteric effects on activity (the different partial activities oftelomerase are described in detail elsewhere in this specification).

[0593] D) Modulator Synthesis

[0594] It is contemplated that the telomerase modulators of theinvention will be made using methods well known in the pharmaceuticalarts, including combinatorial methods and rational drug designtechniques.

[0595] 1) Combinatorial Chemistry Methodology

[0596] The creation and simultaneous screening of large libraries ofsynthetic molecules can be carried out using well-known techniques incombinatorial chemistry, for example, see van Breemen (1997) Anal Chem69:2159-2164; Lam (1997) Anticancer Drug Des 12:145-167 (1997).

[0597] As noted above, combinatorial chemistry methodology can be usedto create vast numbers of oligonucleotides (or other compounds) that canbe rapidly screened for specific oligonucleotides (or compounds) thathave appropriate binding affinities and specificities toward any target,such as the TRT proteins of the invention, can be utilized (for generalbackground information Gold (1995) J. of Biol. Chem. 270:13581-13584).

[0598] 2) Rational Drug Design

[0599] Rational drug design involves an integrated set of methodologiesthat include structural analysis of target molecules, syntheticchemistries, and advanced computational tools. When used to designmodulators, such as antagonists/inhibitors of protein targets, such astelomerase enzyme and hTRT protein, the objective of rational drugdesign is to understand a molecule's three-dimensional shape andchemistry. Rational drug design is aided by X-ray crystallographic dataor NMR data, which can now be determined for the hTRT protein andtelomerase enzyme in accordance with the methods and using the reagentsprovided by the invention. Calculations on electrostatics,hydrophobicities and solvent accessibility is also helpful. See, forexample, Coldren (1997) Proc. Natl. Acad. Sci. USA 94:6635-6640.

[0600] E) Kits

[0601] The invention also provides kits that can be used to aid indetermining whether a test compound is a modulator of a TRT activity.The kit will typically include one or more of the following components:a substantially purified TRT polypeptide or polynucleotide (includingprobes and primers); a plasmid capable of expressing a TRT (e.g., hTRT)when introduced into a cell or cell-free expression system; a plasmidcapable of expressing a TR (e.g., hTR) when introduced into a cell orcell-free expression system; cells or cell lines; a composition todetect a change in TRT activity; and, an instructional material teachinga means to detect and measure a change in the TRT activity, indicatingthat a change in the telomerase activity in the presence of the testcompound is an indicator that the test compound modulates the telomeraseactivity, and one or more containers. The kit can also include means,such as TRAP assay reagents or reagents for a quantitative polymerasechain reaction assay, to measure a change in TRT activity. The kit mayalso include instructional material teaching a means to detect andmeasure a change in the TRT activity, indicating that a change in thetelomerase activity in the presence of the test compound is an indicatorthat the test compound modulates the telomerase activity.

[0602] XI. Transgenic Organisms (Telomerase Knockout Cells and AnimalModels)

[0603] The invention also provides transgenic non-human multicellularorganisms (e.g., plants and non-human animals) or unicellular organisms(e.g., yeast) comprising an exogenous TRT gene sequence, which may be acoding sequence or a regulatory (e.g., promoter) sequence. In oneembodiment, the organism expresses an exogenous TRT polypeptide, havinga sequence of a human TRT protein. In a related embodiment, the organismalso expresses a telomerase RNA component (e.g., hTR).

[0604] The invention also provides unicellular and multicellularorganisms (or cells therefrom) in which at least one gene encoding atelomerase component (e.g., TRT or TR) or telomerase-associated proteinis mutated or deleted (i.e., in a coding or regulatory region) such thatnative telomerase is not expressed, or is expressed at reduced levels orwith different activities when compared to wild-type cells or organisms.Such cells and organisms are often referred to as “gene knock-out” cellsor organisms.

[0605] The invention further provides cells and organisms in which anendogenous telomerase gene (e.g., murine TRT) is either present oroptionally mutated or deleted and an exogenous telomerase gene orvariant (e.g., human TRT) is introduced and expressed. Cells andorganisms of this type will be useful, for example, as model systems foridentifying modulators of hTRT activity or expression; determining theeffects of mutations in telomerase component genes, and other uses suchas determining the developmental timing and tissue location oftelomerase activity (e.g., for assessing when to administer a telomerasemodulator and for assessing any potential side effects).

[0606] Examples of multicellular organisms include plants, insects, andnonhuman animals such as mice, rats, rabbits, monkeys, apes, pigs, andother nonhuman mammals. An example of a unicellular organism is a yeast.

[0607] Methods for alteration or disruption of specific genes (e.g.,endogenous TRT genes) are well known to those of skill, see, e.g.,Baudin et al., 1993, Nucl. Acids Res. 21:3329; Wach et al., 1994, Yeast10:1793; Rothstein, 1991, Methods Enzymol. 194:281; Anderson, 1995,Methods Cell Biol. 48:31; Pettitt et al., 1996, Development122:4149-4157; Ramirez-Solis et al., 1993, Methods Enzymol. 225:855; andThomas et al., 1987, Cell 51:503, each of which is incorporated hereinby reference in its entirety for all purposes.

[0608] The “knockout” cells and animals of the invention include cellsand animals in which one or several units of the endogenous telomeraseenzyme complex have been deleted or inhibited. Reconstitution oftelomerase activity will save the cell or animal from senescence or, forcancer cells, cell death caused by its inability to maintain telomeres.Methods of altering the expression of endogenous genes are well known tothose of skill in the art. Typically, such methods involve altering orreplacing all or a portion of the regulatory sequences controllingexpression of the particular gene to be regulated. The regulatorysequences, e.g., the native promoter can be altered. The conventionaltechnique for targeted mutation of genes involves placing a genomic DNAfragment containing the gene of interest into a vector, followed bycloning of the two genomic arms associated with the targeted gene arounda selectable neomycin-resistance cassette in a vector containingthymidine kinase. This “knock-out” construct is then transfected intothe appropriate host cell, i.e., a mouse embryonic stem (ES) cell, whichis subsequently subjected to positive selection (using G418, forexample, to select for neomycin-resistance) and negative selection(using, for example, FIAU to exclude cells lacking thymidine kinase),allowing the selection of cells which have undergone homologousrecombination with the knockout vector. This approach leads toinactivation of the gene of interest. See, e.g., U.S. Pat. Nos.5,464,764; 5,631,153; 5,487,992; and, 5,627,059.

[0609] “Knocking out” expression of an endogenous gene can also beaccomplished by the use of homologous recombination to introduce aheterologous nucleic acid into the regulatory sequences (e.g., promoter)of the gene of interest. To prevent expression of functional enzyme orproduct, simple mutations that either alter the reading frame or disruptthe promoter can be suitable. To up-regulate expression, a nativepromoter can be substituted with a heterologous promoter that induceshigher levels of transcription. Also, “gene trap insertion” can be usedto disrupt a host gene, and mouse ES cells can be used to produceknockout transgenic animals, as described for example, in Holzschu(1997) Transgenic Res 6: 97-106.

[0610] Altering the expression of endogenous genes by homologousrecombination can also be accomplished by using nucleic acid sequencescomprising the structural gene in question. Upstream sequences areutilized for targeting heterologous recombination constructs. UtilizingTRT structural gene sequence information, such as SEQ ID NO:1, one ofskill in the art can create homologous recombination constructs withonly routine experimentation. Homologous recombination to alterexpression of endogenous genes is described in U.S. Pat. No. 5,272,071,and WO 91/09955, WO 93/09222, WO 96/29411, WO 95/31560, and WO 91/12650.Homologous recombination in mycobacteria is described by Azad (1996)Proc. Natl. Acad. Sci. USA 93:4787; Baulard (1996) J. Bacteriol.178:3091; and Pelicic (1996) Mol. Microbiol. 20:919. Homologousrecombination in animals has been described by Moynahan (1996) Hum. Mol.Genet. 5:875, and in plants by Offringa (1990) EMBO J. 9:3077.

[0611] XII. Glossary

[0612] The following terms are defined infra to provide additionalguidance to one of skill in the practice of the invention: adjuvant,allele (& allelic sequence), amino acids (including hydrophobic, polar,charged), conservative substitution, control elements (& regulatorysequences), derivatized, detectable label, elevated level, epitope,favorable and unfavorable prognosis, fusion protein, gene product, hTR,immortal, immunogen and immunogenic, isolated, modulator, motif, nucleicacid (& polynucleotide), oligonucleotides (& oligomers), operablylinked, polypeptide, probe (including nucleic acid probes & antibodyprobes), recombinant, selection system, sequence, specific binding,stringent hybridization conditions (& stringency), substantial identity(& substantial similarity), substantially pure (& substantiallypurified), telomerase-negative and telomerase-positive cells, telomerasecatalytic activity, telomerase-related, and test compound.

[0613] As used herein, the term “adjuvant” refers to its ordinarymeaning of any substance that enhances the immune response to an antigenwith which it is mixed. Adjuvants useful in the present inventioninclude, but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacteriumparvum are potentially useful adjuvants.

[0614] As used herein, the terms “allele” or “allelic sequence” refer toan alternative form of a nucleic acid sequence (i.e., a nucleic acidencoding hTRT protein). Alleles result from mutations (i.e., changes inthe nucleic acid sequence), and generally produce altered and/ordifferently regulated mRNAs or polypeptides whose structure and/orfunction may or may not be altered. Common mutational changes that giverise to alleles are generally ascribed to natural deletions, additions,or substitutions of nucleotides that may or may not affect the encodedamino acids. Each of these types of changes may occur alone, incombination with the others, or one or more times within a given gene,chromosome or other cellular nucleic acid. Any given gene may have no,one or many allelic forms. As used herein, the term “allele” refers toeither or both a gene or an mRNA transcribed from the gene.

[0615] As used herein, “amino acids” are sometimes specified using thestandard one letter code: Alanine (A), Serine (S), Threonine (T),Aspartic acid (D), Glutamic acid (E) Asparagine (N), Glutamine (Q),Arginine (R), Lysine (K), Isoleucine (I), Leucine (L), Methionine (M),Valine (V), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Proline(P), Glycine (G), Histidine (H), Cysteine (C). Synthetic andnon-naturally occurring amino acid analogues (and/or peptide linkages)are included.

[0616] As used herein, “Hydrophobic amino acids” refers to A, L, I, V,P, F, W, and M. As used herein, “polar amino acids” refers to G, S, T,Y, C, N, and Q. As used herein, “charged amino acids” refers to D, E, H,K, and R.

[0617] As used herein, “conservative substitution”, when describing aprotein refers to a change in the amino acid composition of the proteinthat does not substantially alter the protein's activity. Thus,“conservatively modified variations” of a particular amino acid sequencerefers to amino acid substitutions of those amino acids that are notcritical for protein activity or substitution of amino acids with otheramino acids having similar properties (e.g., acidic, basic, positivelyor negatively charged, polar or non-polar, etc.) such that thesubstitutions of even critical amino acids does not substantially alteractivity. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see also,Creighton (1984) Proteins, W.H. Freeman and Company). One of skill inthe art will appreciate that the above-identified substitutions are notthe only possible conservative substitutions. For example, one mayregard all charged amino acids as conservative substitutions for eachother whether they are positive or negative. In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence can also be “conservatively modified variations”. One can alsomake a “conservative substitution” in a recombinant protein by utilizingone or more codons that differ from the codons employed by the native orwild-type gene. In this instance, a conservative substitution alsoincludes substituting a codon for an amino acid with a different codonfor the same amino acid.

[0618] As used herein, “control elements” or “regulatory sequences”include enhancers, promoters, transcription terminators, origins ofreplication, chromosomal integration sequences, 5′ and 3′untranslatedregions, with which proteins or other biomolecules interact to carry outtranscription and translation. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer, e.g.,derived from immunoglobulin genes, SV40, cytomegalovirus, and apolyadenylation sequence, and may include splice donor and acceptorsequences. Depending on the vector system and host utilized, any numberof suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used

[0619] As used herein, a “derivatized” polynucleotide, oligonucleotide,or nucleic acid refers to oligo- and polynucleotides that comprise aderivatized substituent. In some embodiments, the substituent issubstantially non-interfering with respect to hybridization tocomplementary polynucleotides. Derivatized oligo- or polynucleotidesthat have been modified with appended chemical substituents (e.g., bymodification of an already synthesized oligo- or poly-nucleotide, or byincorporation of a modified base or backbone analog during synthesis)may be introduced into a metabolically active eukaryotic cell tohybridize with an hTRT DNA, RNA, or protein where they produce analteration or chemical modification to a local DNA, RNA, or protein.Alternatively, the derivatized oligo or polynucleotides may interactwith and alter hTRT polypeptides, telomerase-associated proteins, orother factors that interact with hTRT DNA or hTRT gene products, oralter or modulate expression or function of hTRT DNA, RNA or protein.Illustrative attached chemical substituents include: europium (III)texaphyrin, cross-linking agents, psoralen, metal chelates (e.g.,iron/EDTA chelate for iron catalyzed cleavage), topoisomerases,endonucleases, exonucleases, ligases, phosphodiesterases, photodynamicporphyrins, chemotherapeutic drugs (e.g., adriamycin, doxirubicin),intercalating agents, base-modification agents, immunoglobulin chains,and oligonucleotides. Iron/EDTA chelates are chemical substituents oftenused where local cleavage of a polynucleotide sequence is desired(Hertzberg et al., 1982, J. Am. Chem. Soc. 104: 313; Hertzberg andDervan, 1984, Biochemistry 23: 3934; Taylor et al., 1984, Tetrahedron40: 457; Dervan, 1986, Science 232: 464. Illustrative attachmentchemistries include: direct linkage, e.g., via an appended reactiveamino group (Corey and Schultz (1988) Science 238: 1401, which isincorporated herein by reference) and other direct linkage chemistries,although streptavidin/biotin and digoxigenin/anti-digoxigenin antibodylinkage methods can also be used. Methods for linking chemicalsubstituents are provided in U.S. Pat. Nos. 5,135,720, 5,093,245, and5,055,556, which are incorporated herein by reference. Other linkagechemistries may be used at the discretion of the practitioner.

[0620] As used herein, a “detectable label” has the ordinary meaning inthe art and refers to an atom (e.g., radionuclide), molecule (e.g.,fluorescein), or complex, that is or can be used to detect (e.g., due toa physical or chemical property), indicate the presence of a molecule orto enable binding of another molecule to which it is covalently bound orotherwise associated. The term “label” also refers to covalently boundor otherwise associated molecules (e.g., a biomolecule such as anenzyme) that act on a substrate to produce a detectable atom, moleculeor complex. Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Labels useful in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, greenfluorescent protein, enhanced green fluorescent protein, lissamine,phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluor X [Amersham],SyBR Green I & II [Molecular Probes], and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., hydrolases, particularlyphosphatases such as alkaline phosphatase, esterases and glycosidases,or oxidoreductases, particularly peroxidases such as horse radishperoxidase, and others commonly used in ELISAs), substrates, cofactors,inhibitors, chemiluminescent groups, chromogenic agents, andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means ofdetecting such labels are well known to those of skill in the art. Thus,for example, radiolabels and chemiluminescent labels may be detectedusing photographic film or scintillation counters, fluorescent markersmay be detected using a photodetector to detect emitted light (e.g., asin fluorescence-activated cell sorting). Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate,and colorimetric labels are detected by simply visualizing the coloredlabel. Thus, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. The label may be coupled directly or indirectly to thedesired component of the assay according to methods well known in theart. Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal generating system, such as a detectable enzyme, a fluorescentcompound, or a chemiluminescent compound. A number of ligands andanti-ligands can be used. Where a ligand has a natural anti-ligand, forexample, biotin, thyroxine, and cortisol, it can be used in conjunctionwith the labeled, naturally occurring anti-ligands. Alternatively, anyhaptenic or antigenic compound can be used in combination with anantibody. The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Means of detecting labels are well known to those of skillin the art. Thus, for example, where the label is a radioactive label,means for detection include a scintillation counter, photographic filmas in autoradiography, or storage phosphor imaging. Where the label is afluorescent label, it may be detected by exciting the fluorochrome withthe appropriate wavelength of light and detecting the resultingfluorescence. The fluorescence may be detected visually, by means ofphotographic film, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Similarly,enzymatic labels may be detected by providing the appropriate substratesfor the enzyme and detecting the resulting reaction product. Also,simple colorimetric labels may be detected by observing the colorassociated with the label. It will be appreciated that when pairs offluorophores are used in an assay, it is often preferred that the theyhave distinct emission patterns (wavelengths) so that they can be easilydistinguished.

[0621] The phrase “elevated level” refers to an amount of hTRT geneproduct (or other specified substance or activity) in a cell that iselevated or higher than the level in a reference standard, e.g., fordiagnosis, the level in normal, telomerase-negative cells in anindividual or in other individuals not suffering from the condition, andfor prognosis, the level in tumor cells from a variety of grades orclasses of, e.g., tumors.

[0622] As used herein, the term “epitope” has its ordinary meaning of asite on an antigen recognized by an antibody. Epitopes are typicallysegments of amino acids which are a small portion of the whole protein.Epitopes may be conformational (i.e., discontinuous). That is, they maybe formed from amino acids encoded by noncontiguous parts of a primarysequence that have been juxtaposed by protein folding.

[0623] The terms “favorable prognosis” and “unfavorable prognosis” areknown in the art. In the context of cancers, “favorable prognosis” meansthat there is a likelihood of tumor regression or longer survival timesfor patients with a favorable prognosis relative to those withunfavorable prognosis, whereas “unfavorable prognosis” means that thetumor is likely to be more aggressive, i.e., grow faster and/ormetastasize, resulting in a poor outcome or a more rapid course ofdisease progression for the patient.

[0624] As used herein, the term “fusion protein,” refers to a compositeprotein, i.e., a single contiguous amino acid sequence, made up of two(or more) distinct, heterologous polypeptides which are not normallyfused together in a single amino acid sequence. Thus, a fusion proteinmay include a single amino acid sequence that contains two entirelydistinct amino acid sequences or two similar or identical polypeptidesequences, provided that these sequences are not normally found togetherin the same configuration in a single amino acid sequence found innature. Fusion proteins may generally be prepared using eitherrecombinant nucleic acid methods, i.e., as a result of transcription andtranslation of a recombinant gene fusion product, which fusion comprisesa segment encoding a polypeptide of the invention and a segment encodinga heterologous protein, or by chemical synthesis methods well known inthe art. The non-hTRT region(s) of the fusion protein can be fused tothe amino terminus of the hTRT polypeptide or the carboxyl terminus, orboth or the non-hTRT region can be inserted into the interior of theprotein sequence (by moiety inserting or by replacing amino acids) orcombinations of the foregoing can be performed.

[0625] As used herein, the term “gene product” refers to an RNA moleculetranscribed from a gene, or a protein encoded by the gene or translatedfrom the RNA.

[0626] As used herein, “hTR” (human telomerase RNA) refers to the RNAcomponent of human telomerase and any naturally occurring alleles andvariants or recombinant variants. hTR is described in detail in U.S.Pat. No. 5,583,016 which is incorporated herein by reference in itsentirety and for all purposes.

[0627] As used herein, the term “immortal,” when referring to a cell,has its normal meaning in the telomerase art and refers to cells thathave apparently unlimited replicative potential. Immortal can also referto cells with increased proliferative capacity relative to theirunmodified counterparts. Examples of immortal human cells are malignanttumor cells, germ line cells, and certain transformed human cell linescultured in vitro (e.g., cells that have become immortal followingtransformation by viral oncogenes or otherwise). In contrast, mostnormal human somatic cells are mortal, i.e., have limited replicativepotential and become senescent after a finite number of cell divisions.

[0628] As used herein, the terms “immunogen” and “immunogenic” havetheir ordinary meaning in the art, i.e, an immunogen is a molecule, suchas a protein or other antigen, that can elicit an adaptive immuneresponse upon injection into a person or an animal.

[0629] As used herein, “isolated,” when referring to a molecule orcomposition, such as, for example, an RNP (e.g., at least one proteinand at least one RNA), means that the molecule or composition isseparated from at least one other compound, such as a protein, otherRNAs, or other contaminants with which it is associated in vivo or inits naturally occurring state. Thus, an RNP is considered isolated whenthe RNP has been isolated from any other component with which it isnaturally associated, e.g., cell membrane, as in a cell extract. Anisolated composition can, however, also be substantially pure.

[0630] As used herein, “modulator” refers to any synthetic or naturalcompound or composition that can change in any way either or both the“full” or any “partial activity” of a telomerase reverse transcriptase(TRT). A modulator can be an agonist or an antagonist. A modulator canbe any organic and inorganic compound; including, but not limited to,for example, small molecules, peptides, proteins, sugars, nucleic acids,fatty acids and the like.

[0631] As used herein, “motif” refers to a sequence of contiguous aminoacids (or to a nucleic acid sequence that encodes a sequence ofcontiguous amino acids) that defines a feature or structure in a proteinthat is common to or conserved in all proteins of a defined class ortype. The motif or consensus sequence may include both conserved andnon-conserved residues. The conserved residues in the motif sequenceindicate that the conserved residue or class (i.e., hydrophobic, polar,non-polar, or other class) of residues is typically present at theindicated location in each protein (or gene or mRNA) of the class ofproteins defined by the motif. Motifs can differ in accordance with theclass of proteins. Thus, for example, the reverse transcriptase enzymesform a class of proteins than can be defined by one or more motifs, andthis class includes telomerase enzymes. However, the telomerase enzymescan also be defined as the class of enzymes with motifs characteristicfor that class. Those of skill recognize that the identification of aresidue as a conserved residue in a motif does not mean that everymember of the class defined by the motif has the indicated residue (orclass of residues) at the indicated position, and that one or moremembers of the class may have a different residue at the conservedposition.

[0632] As used herein, the terms “nucleic acid” and “polynucleotide” areused interchangeably. Use of the term “polynucleotide” is not intendedto exclude oligonucleotides (i.e., short polynucleotides) and can alsorefer to synthetic and/or non-naturally occurring nucleic acids (i.e.,comprising nucleic acid analogues or modified backbone residues orlinkages).

[0633] As used herein “oligonucleotides” or “oligomers” refer to anucleic acid sequence of approximately 7 nucleotides or greater, and asmany as approximately 100 nucleotides, which can be used as a primer,probe or amplimer. Oligonucleotides are often between about 10 and about50 nucleotides in length, more often between about 14 and about 35nucleotides, very often between about 15 and about 25 nucleotides, andthe terms oligonucleotides or oligomers can also refer to syntheticand/or non-naturally occurring nucleic acids (i.e., comprising nucleicacid analogues or modified backbone residues or linkages).

[0634] As used herein, the term “operably linked,” refers to afunctional relationship between two or more nucleic acid (e.g., DNA)segments: for example, a promoter or enhancer is operably linked to acoding sequence if it stimulates the transcription of the sequence in anappropriate host cell or other expression system. Generally, sequencesthat are operably linked are contiguous, and in the case of a signalsequence both contiguous and in reading phase. However, enhancers neednot be located in close proximity to the coding sequences whosetranscription they enhance.

[0635] As used herein, the term “polypeptide” is used interchangeablyherein with the term “protein,” and refers to a polymer composed ofamino acid residues linked by amide linkages, including synthetic,naturally-occurring and non-naturally occurring analogs thereof (aminoacids and linkages). Peptides are examples of polypeptides.

[0636] As used herein, a “probe” refers to a molecule that specificallybinds another molecule. One example of a probe is a “nucleic acid probe”that specifically binds (i.e., anneals or hybridizes) to a substantiallycomplementary nucleic acid. Another example of a probe is an “antibodyprobe” that specifically binds to a corresponding antigen or epitope.

[0637] As used herein, “recombinant” refers to a polynucleotidesynthesized or otherwise manipulated in vitro (e.g., “recombinantpolynucleotide”), to methods of using recombinant polynucleotides toproduce gene products in cells or other biological systems, or to apolypeptide (“recombinant protein”) encoded by a recombinantpolynucleotide.

[0638] As used herein, a “selection system,” in the context of stablytransformed cell lines, refers to a method for identifying and/orselecting cells containing a recombinant nucleic acid of interest. Alarge variety of selection systems are known for identification oftransformed cells and are suitable for use with the present invention.For example, cells transformed by plasmids or other vectors can beselected by resistance to antibiotics conferred by genes contained onthe plasmids, such as the well known amp, gpt, neo and hyg genes, orother genes such as the herpes simplex virus thymidine kinase (Wigler etal., Cell 11:223-32 [1977]) and adenine phosphoribosyltransferase (Lowyet al., Cell 22:817 [1980]) genes which can be employed in tk− or aprt−cells, respectively. Also, antimetabolite, antibiotic or herbicideresistance can be used as the basis for selection; for example, dhfrwhich confers resistance to methotrexate and is also useful for geneamplification (Wigler et al., Proc. Natl. Acad. Sci., 77:3567 [1980]);npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin et al., J. Mol. Biol., 150:1 [1981]) and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, in McGraw Hill Yearbook ofScience and Technology, McGraw Hill, New York N.Y., pp 191-196, [1992]).Additional selectable genes have been described, for example, hygromycinresistance-conferring genes, trpB, which allows cells to utilize indolein place of tryptophan, or hisD, which allows cells to utilize histinolin place of histidine (Hartman and Mulligan, Proc. Natl. Acad. Sci.,85:8047 [1988]). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, beta-glucuronidase and itssubstrate, GUS, and luciferase and its substrate, luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Meth. Mol. Biol., 55:121 [1995]).

[0639] As used herein, the “sequence” of a gene (unless specificallystated otherwise), nucleic acid, protein, or peptide refers to the orderof nucleotides in either or both strands of a double-stranded DNAmolecule, e.g., the sequence of both the coding strand and itscomplement, or of a single-stranded nucleic acid molecule, or to theorder of amino acids in a peptide or protein.

[0640] As used herein, “specific binding” refers to the ability of onemolecule, typically an antibody or polynucleotide, to contact andassociate with another specific molecule even in the presence of manyother diverse molecules. For example, a single-stranded polynucleotidecan specifically bind to a single-stranded polynucleotide that iscomplementary in sequence, and an antibody specifically binds to (or “isspecifically immunoreactive with”) its corresponding antigen.

[0641] As used herein, “stringent hybridization conditions” or“stringency” refers to conditions in a range from about 5° C. to about20° C. or 25° C. below the melting temperature (T_(m)) of the targetsequence and a probe with exact or nearly exact complementarity to thetarget. As used herein, the melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf-dissociated into single strands. Methods for calculating the T_(m)of nucleic acids are well known in the art (see, e.g., Berger and Kimmel(1987) METHODS IN ENZYMOLOGY, VOL.152: GUIDE TO MOLECULAR CLONINGTECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989)MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold SpringHarbor Laboratory hereinafter, “Sambrook”), both incorporated herein byreference). As indicated by standard references, a simple estimate ofthe T_(m) value may be calculated by the equation: T_(m)=81.5+0.41(%G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g.,Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACIDHYBRIDIZATION (1985)). Other references include more sophisticatedcomputations which take structural as well as sequence characteristicsinto account for the calculation of T_(m). The melting temperature of ahybrid (and thus the conditions for stringent hybridization) is affectedby various factors such as the length and nature (DNA, RNA, basecomposition) of the probe and nature of the target (DNA, RNA, basecomposition, present in solution or immobilized, and the like), and theconcentration of salts and other components (e.g., the presence orabsence of formamide, dextran sulfate, polyethylene glycol). The effectsof these factors are well known and are discussed in standard referencesin the art, e.g., Sambrook, supra and Ausubel et al. supra. Typically,stringent hybridization conditions are salt concentrations less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH7.0 to 8.3, and temperatures at least about 30° C. for short probes(e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g., greater than 50 nucleotides). As noted, stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide, in which case lower temperatures may be employed.

[0642] As used herein, the term “substantial identity,” “substantialsequence identity,” or “substantial similarity” in the context ofnucleic acids, refers to a measure of sequence similarity between twopolynucleotides. Substantial sequence identity can be determined byhybridization under stringent conditions, by direct comparison, or othermeans. For example, two polynucleotides can be identified as havingsubstantial sequence identity if they are capable of specificallyhybridizing to each other under stringent hybridization conditions.Other degrees of sequence identity (e.g., less than “substantial”) canbe characterized by hybridization under different conditions ofstringency. Alternatively, substantial sequence identity can bedescribed as a percentage identity between two nucleotide (orpolypeptide) sequences. Two sequences are considered substantiallyidentical when they are at least about 60% identical, preferably atleast about 70% identical, or at least about 80% identical, or at leastabout 90% identical, or at least about 95% or 98% to 100% identical.Percentage sequence (nucleotide or amino acid) identity is typicallycalculated by determining the optimal alignment between two sequencesand comparing the two sequences. For example an exogenous transcriptused for protein expression can be described as having a certainpercentage of identity or similarity compared to a reference sequence(e.g., the corresponding endogenous sequence). Optimal alignment ofsequences may be conducted using the local homology algorithm of Smithand Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by thesearch for similarity method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. U.S.A. 85: 2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by inspection. The best alignment (i.e., resulting in thehighest percentage of identity) generated by the various methods isselected. Typically these algorithms compare the two sequences over a“comparison window” (usually at least 18 nucleotides in length) toidentify and compare local regions of sequence similarity, thus allowingfor small additions or deletions (i.e., gaps). Additions and deletionsare typically 20 percent or less of the length of the sequence relativeto the reference sequence, which does not comprise additions ordeletions. It is sometimes desirable to describe sequence identitybetween two sequences in reference to a particular length or region(e.g., two sequences may be described as having at least 95% identityover a length of at least 500 basepairs). Usually the length will be atleast about 50, 100, 200, 300, 400 or 500 basepairs, amino acids, orother residues. The percentage of sequence identity is calculated bycomparing two optimally aligned sequences over the region of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, or U) occurs in both sequences to yield thenumber of matched positions, and determining the number (or percentage)of matched positions as compared to the total number of bases in thereference sequence or region of comparison. An additional algorithm thatis suitable for determining sequence similarity is the BLAST algorithm,which is described in Altschul (1990) J. Mol. Biol. 215: 403-410; andShpaer (1996) Genomics 38:179-191. Software for performing BLASTanalyses is publicly available at the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence that either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al, supra.). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad.Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10,M=5, N=−4, and a comparison of both strands. The term BLAST refers tothe BLAST algorithm which performs a statistical analysis of thesimilarity between two sequences; see, e.g., Karlin (1993) Proc. Natl.Acad. Sci. USA 90:5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid can be considered similar to a TRT nucleic acid if the smallest sumprobability in a comparison of the test nucleic acid to an TRT nucleicacid is less than about 0.5, 0.2, 0.1, 0.01, or 0.001. Alternatively,another indication that two nucleic acid sequences are similar is thatthe polypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.It will be recognized that homologous non-human TRT polynucleotides mayhave less that “substantial” nucleotide identity in certain regions, asthe term “substantial identity” is defined herein. For example, EuplotesTRT is substantially less than about 60% identical to the hTRTpolynucleotide of SEQ ID NO:1 in certain regions, although the two genesare homologs.

[0643] As used herein, the terms “substantial identity,” “substantialsequence identity,”or “substantial similarity” in the context of apolypeptide, refers to a degree of similarity between two polypeptidesin which a polypeptides comprises a sequence with at least 70% sequenceidentity to a reference sequence, or 80%, or 85% or up to 100% sequenceidentity to the reference sequence, or most preferably 90% identity overa comparison window of about 10-20 amino acid residues. Amino acidsequence similarity, or sequence identity, is determined by optimizingresidue matches, if necessary, by introducing gaps as required. SeeNeedleham et al. (1970) J. Mol. Biol. 48: 443-453; and Sankoff et al.,1983, Time Warps, String Edits, and Macromolecules, The Theory andPractice of Sequence Comparison, Chapter One, Addison-Wesley, Reading,Mass.; and software packages from IntelliGenetics, Mountain View,Calif., and the University of Wisconsin Genetics Computer Group,Madison, Wis. As will be apparent to one of skill, the terms“substantial identity”, “substantial similarity” and “substantialsequence identity” can be used interchangeably with regard topolypeptides or polynucleotides. It will be recognized that homologousnon-human TRT polypeptides may have less that “substantial” sequenceidentity in certain regions, as the term “substantial identity” isdefined herein. For example, Euplotes TRT protein is substantially lessthan about 60% identical to the hTRT polynucleotide of SEQ ID NO:2 incertain regions, although the two genes are homologs. In the context ofTRT polypeptides from different species, for example, “significanthomology” at the amino acid sequence means at least about 20% sequenceidentity in region of about 20 to about 40 residues, or at least about40% sequence identity in region of at least about 20% sequence identity.

[0644] As used herein, the term “substantially pure,” or “substantiallypurified,” when referring to a composition comprising a specifiedreagent, such as an antibody (e.g. an anti-hTRT antibody), means thatthe specified reagent is at least about 75%, or at least about 90%, orat least about 95%, or at least about 99% or more of the composition(not including, e.g., solvent or buffer). Thus, for example, a preferredimmunoglobulin preparation of the invention that specifically binds anhTRT polypeptide is substantially purified.

[0645] As used herein, a “telomerase negative” cell is one in whichtelomerase is not expressed, i.e., no telomerase catalytic activity canbe detected using a conventional assay or a TRAP assay for telomerasecatalytic activity. As used herein, a “telomerase positive” cell is acell in which telomerase is expressed (i.e. telomerase activity can bedetected).

[0646] As used herein, a “telomerase-related” disease or condition is adisease or condition in a subject that is correlated with an abnormallyhigh level of telomerase activity in cells of the individual, which caninclude any telomerase activity at all for most normal somatic cells, orwhich is correlated with a low level of telomerase activity that resultsin impairment of a normal cell function. Examples of telomerase-relatedconditions include, e.g., cancer (high telomerase activity in malignantcells) and infertility (low telomerase activity in germ-line cells).

[0647] As used herein, “test compound” or “agent” refers to anysynthetic or natural compound or composition. The term includes allorganic and inorganic compounds; including, for example, smallmolecules, peptides, proteins, sugars, nucleic acids, fatty acids andthe like.

XIII. EXAMPLES

[0648] The following examples are provided to illustrate the presentinvention, and not by way of limitation.

[0649] In the following sections, the following abbreviations apply: eq(equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); l or L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); ° C. (degrees Centigrade); RPN(ribonucleoprotein); mreN (2′-O-methylribonucleotides); dNTP(deoxyribonucleotide); dH₂O (distilled water); DDT (dithiothreitol);PMSF (phenylmethylsulfonyl fluoride); TE (10 mM Tris HCl, 1 mM EDTA,approximately pH 7.2); KGlu (potassium glutamate); SSC (salt and sodiumcitrate buffer); SDS (sodium dodecyl sulfate); PAGE (polyacrylamide gelelectrophoresis); Novex (Novex, San Diego, Calif.); BioRad (Bio-RadLaboratories, Hercules, Calif.); Pharmacia (Pharmacia Biotech,Piscataway, N.J.); Boehringer-Mannheim (Boehringer-Mannheim Corp.,Concord, Calif.); Amersham (Amersham, Inc., Chicago, Ill.); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); NEB (New EnglandBiolabs, Beverly, Mass.); Pierce (Pierce Chemical Co., Rockford, Ill.);Beckman (Beckman Instruments, Fullerton, Calif.); Lab Industries (LabIndustries, Inc., Berkeley, Calif.); Eppendorf (Eppendorf Scientific,Madison, Wis.); and Molecular Dynamics (Molecular Dynamics, Sunnyvale,Calif.).

Example 1 Isolation of Telomerase Proteins and Clones

[0650] The following example details the isolation of telomeraseproteins and clones from various organisms, including the euplotes p.123, hTRT, TRT and S. pombe TRT telomerase cDNA clones.

[0651] A. Background

[0652] i) Introduction

[0653] This section provides an overview of the purification and cloningof TRT genes, which is described in greater detail in subsequentsections of this Example. While telomerase RNA subunits have beenidentified in ciliates, yeast and mammals, protein subunits of theenzyme have not been identified as such prior to the present invention.Purification of telomerase from the ciliated protozoan Euplotesaediculatus yielded two proteins, termed p123 and p43 (see infra;Lingner (1996) Proc. Natl. Acad. Sci. U.S.A. 93:10712). Euplotesaediculatus is a hypotrichous ciliate having a macronucleus containingabout 8×10⁷ telomeres and about 3×10⁵ molecules of telomerase. Afterpurification, the active telomerase complex had a molecular mass ofabout 230 kD, corresponding to a 66 kD RNA subunit and two proteins ofabout 123 kD and 43 kD (Lingner (1996) supra). Photocross-linkingexperiments indicated that the larger p123 protein was involved inspecific binding of the telomeric DNA substrate (Lingner, (1996) supra).

[0654] The p123 and p43 proteins were sequenced and the cDNA cloneswhich encoded these proteins were isolated. These Euplotes sequenceswere found to be unrelated to the Tetrahymena telomerase-associatedproteins p80 and p95. Sequence analysis of the Euplotes p123 revealedreverse transcriptase (RT) motifs. Furthermore, sequence analysis of theEuplotes p123 by comparison to other sequences revealed a yeast homolog,termed Est2 protein (Lingner (1997) Science 276:561). Yeast Est2 hadpreviously been shown to be essential for telomere maintenance in vivo(Lendvay (1996) Genetics 144:1399) but had not been identified as atelomerase catalytic protein. Site-specific mutagenesis demonstratedthat the RT motifs of yeast Est2 are essential for telomeric DNAsynthesis in vivo and in vitro (Lingner (1997) supra).

[0655] ii) Identifying and Characterizing S. pombe Telomerase

[0656] PCR amplification of S. pombe DNA was carried out with degeneratesequence primers designed from the Euplotes p123 RT motifs as describedbelow. Of the four prominent PCR products generated, a 120 base pairband encoded a peptide sequence homologous to p123 and Est2. This PCRproduct was used as a probe in colony hybridization and identified twooverlapping clones from an S. pombe genomic library and three from an S.pombe cDNA library. Sequence analysis revealed that none of the three S.pombe cDNA clones was full length, so RT-PCR was used to obtain thesequences encoding the protein's N-terminus.

[0657] Complete sequencing of these clones revealed a putative S. pombetelomerase RT gene, trt1. The complete nucleotide sequence of trt1 hasbeen deposited in GenBank, accession number AF015783 (see FIG. 15).

[0658] To test S. pombe trt1 (as a catalytic subunit, two deletionconstructs were created. Analysis of the sequence showed that trt1encoded a basic protein with a predicted molecular mass of 116 kD. Itwas found that homology with p123 and Est2 was especially high in theseven reverse transcriptase motifs, underlined and designated as motifs1, 2, A, B, C, D, and E (see FIG. 63). An additional telomerase-specificmotif, designated the T-motif, was also found. Fifteen introns, rangingin size from 36 to 71 base pairs, interrupted the coding sequence.

[0659] To test S. pombe trt1 as a catalytic subunit, two deletionconstructs were created. One removed only motifs B through D in the RTdomains. The second removed 99% of the open reading frame.

[0660] Haploid cells grown from S. pombe spores of both mutants showedprogressive telomere shortening to the point where hybridization totelomeric repeats became almost undetectable. A trt1⁺/trt1⁻ diploid wassporulated and the resulting tetrads were dissected and germinated on ayeast extract medium supplemented with amino acids (a YES plate, Alfa(1993) Experiments with Fission Yeast, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). Colonies derived from each spore weregrown at 32° C. for three days, and streaked successively to fresh YESplates every three days. A colony from each round was placed in six mlof YES liquid culture at 32° C. and grown to stationary phase. GenomicDNA was prepared. After digestion with ApaI, DNA was subjected toelectrophoresis on a 2.3% agarose gel, stained with ethidium bromide toconfirm approximately equal loading in each lane, then transferred to anylon membrane and hybridized to a telomeric DNA probe.

[0661] Senescence was indicated by the delayed onset of growth orfailure to grow on agar (typically at the fourth streak-out aftergermination) and by colonies with increasingly ragged edges (colonymorphology shown in FIG. 22C) and by increasingly high fractions ofelongated cells (as shown in FIG. 22D). Cells were plated on MinimalMedium (Alfa (1993) supra) with glutamic acid substituted for ammoniumchloride for two days at 32° C. prior to photography.

[0662] When individual enlarged cells were separated on the dissectingmicroscope, the majority were found to undergo no further division. Thesame telomerase negative (trt1⁻) cell population always containednormal-sized cells which continued to divide, but which frequentlyproduced non-dividing progeny. The telomerase-negative survivors may usea recombinational mode of telomere maintenance as documented in buddingyeast strains that have various telomere-replication genes deleted(Lendvay (1996) supra, Lundblad (1993) Cell 73:347).

[0663] iii) Identifying and Characterizing Human Telomerase

[0664] An EST (expressed sequence tag) derived from human telomerasereverse transcriptase (hTRT) cDNA was identified by a BLAST search ofthe dbEST (expressed sequence tag) Genbank database using the Euplotes123 kDa peptide and nucleic acid sequences, as well as theSchizosaccharomyces protein and corresponding cDNA (tez1) sequences. TheEST, designated Genbank AA28196, is 389 nucleotides long and itcorresponds to positions 1679 to 2076 of clone 712562 (FIG. 18), wasobtained from the I.M.A.G.E. Consortium (Human Genome Center, DOE,Lawrence Livermore National Laboratory, Livermore, Calif.). This clonewas obtained from a cDNA library of germinal B cells derived by flowsorting of tonsil cells. Complete sequencing of this hTRT cDNA cloneshowed all eight telomerase RT (TRT) motifs. However, this hTRT clonedid not encode a contiguous portion of a TRT because RT motifs B′, C, D,and E, were contained in a different open reading frame than the moreN-terminal RT motifs. In addition, the distance between RT motifs A andB was substantially shorter than that of the three previously known(non-human) TRTs.

[0665] To isolate a full length cDNA clone, a cDNA library derived formthe human 293 cell line (described above) which expresses high levels oftelomerase activity, was screened. A lambda cDNA library from the 293cell line was partitioned into 25 pools containing about 200,000 plaqueseach. Each pool was screened by PCR with the primer pair5′-CGGAAGAGTGTCTGGAGCAA-3′ (SEQ ID NO:551) and 5′-GGATGAAGCGGAGTCTGGA-3′(SEQ ID NO:459). Six subpools of one positive primary pool were furtherscreened by PCR using this same primer pair. For both the primary andthe secondary subpool screening, hTRT was amplified for a total of 31cycles at: 94° C., 45 seconds; 60° C., 45 seconds; and 72° C., 90seconds. As a control, RNA of the house-keeping enzyme GAPDH wasamplified using the primer pair 5′-CTCAGACACCATGGGGAAGGTGA-3′ (SEQ IDNO:552) and 5′-ATGATCTTGAGGCTGTTGTCATA-3′ (SEQ ID NO:553) for a total of16 cycles at 94° C., 45 seconds; 55° C., 45 seconds; and 72° C., 90seconds.

[0666] One hTRT positive subpool from the secondary screening was thenscreened by plaque hybridization with a probe from the 5′ region ofclone #712562. One phage was positively identified (designated Lambdaphage 25-1.1, ATCC 209024, deposited May 12, 1997). It contained anapproximately four kilobase insert, which was excised and subcloned intothe EcoRI site of pBluescript II SK+ vector (Stratagene, San Diego,Calif.) as an EcoRI fragment. This cDNA clone-containing plasmid wasdesignated pGRN121. The cDNA insert totals approximately 4kilobasepairs. The complete nucleotide sequence of the human hTRT cDNA(pGRN121) has been deposited in Genbank (accession AF015950) and theplasmid has been deposited with the ATCC (ATCC 209016, deposited May 6,1997).

[0667] B. Growth of Euplotes aediculatus

[0668] In this Example, cultures of E. aediculatus were obtained fromDr. David Prescott, MCDB, University of Colorado. Dr. Prescottoriginally isolated this culture from pond water, although this organismis also available from the ATCC (ATCC #30859). Cultures were grown asdescribed by Swanton et al., (Swanton et al., Chromosoma 77:203 [1980]),under non-sterile conditions, in 15-liter glass containers containingChlorogonium as a food source. Organisms were harvested from thecultures when the density reached approximately 10⁴ cells/ml.

[0669] C. Preparation of Nuclear Extracts

[0670] In this Example, nuclear extracts of E. aediculatus were preparedusing the method of Lingner et al., (Lingner et al., Genes Develop.,8:1984 [1994]), with minor modifications, as indicated below. Briefly,cells grown as described in Part B were concentrated with 15 μm Nytexfilters and cooled on ice. The cell pellet was resuspended in a finalvolume of 110 ml TMS/PMSF/spermidine phosphate buffer. The stockTMS/PMSF/spermidine phosphate buffer was prepared by adding 0.075 gspermidine phosphate (USB) and 0.75 ml PMSF (from 100 mM stock preparedin ethanol) to 150 ml TMS. TMS comprised 10 mM Tris-acetate, 10 mMMgCl₂, 85.5752 g sucrose/liter, and 0.33297 g CaCl₂/liter, pH 7.5.

[0671] After resuspension in TMS/PMSF/spermidine phosphate buffer, 8.8ml 10% NP-40 and 94.1 g sucrose were added and the mixture placed in asiliconized glass beaker with a stainless steel stirring rod attached toan overhead motor. The mixture was stirred until the cells werecompletely lysed (approximately 20 minutes). The mixture was thencentrifuged for 10 minutes at 7500 rpm (8950×g), at 4EC, using a BeckmanJS-13 swing-out rotor. The supernatant was removed and nuclei pellet wasresuspended in TMS/PMSF/spermidine phosphate buffer, and centrifugedagain, for 5 minutes at 7500 rpm (8950×g), at 4EC, using a Beckman JS-13swing-out rotor.

[0672] The supernatant was removed and the nuclei pellet was resuspendedin a buffer comprised of 50 mM Tris-acetate, 10 mM MgCl₂, 10% glycerol,0.1% NP-40, 0.4 M KGlu, 0.5 mM PMSF, pH 7.5, at a volume of 0.5 mlbuffer per 10 g of harvested cells. The resuspended nuclei were thendounced in a glass homogenizer with approximately 50 strokes, and thencentrifuged for 25 minutes at 14,000 rpm at 4° C., in an Eppendorfcentrifuge. The supernatant containing the nuclear extract wascollected, frozen in liquid nitrogen, and stored at −80° C. until used.

[0673] D. Purification of Telomerase

[0674] In this Example, nuclear extracts prepared as described in Part Cwere used to purify E. aediculatus telomerase. In this purificationprotocol, telomerase was first enriched by chromatography on anAffi-Gel-heparin column, and then extensively purified by affinitypurification with an antisense oligonucleotide. As the template regionof telomerase RNA is accessible to hybridization in the telomerase RNPparticle, an antisense oligonucleotide (i.e., the “affinityoligonucleotide”) was synthesized that was complementary to thistemplate region as an affinity bait for the telomerase. A biotin residuewas included at the 5′ end of the oligonucleotide to immobilize it to anavidin column.

[0675] Following the binding of the telomerase to the oligonucleotide,and extensive washing, the telomerase was eluted by use of adisplacement oligonucleotide. The affinity oligonucleotide included DNAbases that were not complementary to the telomerase RNA 5′ to thetelomerase-specific sequence. As the displacement oligonucleotide wascomplementary to the affinity oligonucleotide for its entire length, itwas able to form a more thermodynamically stable duplex than thetelomerase bound to the affinity oligonucleotide. Thus, addition of thedisplacement oligonucleotide resulted in the elution of the telomerasefrom the column.

[0676] The nuclear extracts prepared from 45 liter cultures were frozenuntil a total of 34 ml of nuclear extract was collected. Thiscorresponded to 630 liters of culture (i.e., approximately 4×10⁹ cells).The nuclear extract was diluted with a buffer to 410 ml, to providefinal concentrations of 20 mM Tris-acetate, 1 mM MgCl₂, 0.1 mM EDTA, 33mM KGlu, 10% (vol/vol) glycerol, 1 mM dithiothreitol (DTT), and 0.5 mMphenylmethylsulfonyl fluoride (PMSF), at a pH of 7.5.

[0677] The diluted nuclear extract was applied to an Affi-Gel-heparingel column (Bio-Rad), with a 230 ml bed volume and 5 cm diameter,equilibrated in the same buffer and eluted with a 2-liter gradient from33 to 450 mM KGlu. The column was run at 4° C., at a flow rate of 1column volume/hour. Fractions of 50 mls each were collected and assayedfor telomerase activity as described in Part E. Telomerase was elutedfrom the column at approximately 170 mM KGlu. Fractions containingtelomerase (approximately 440 ml) were pooled and adjusted to 20 mMTris-acetate, 10 mM MgCl₂, 1 mM EDTA, 300 mM KGlu, 10% glycerol, 1 mMDTT, and 1% Nonidet P-40. This buffer was designated as “WB.”

[0678] To this preparation, 1.5 nmol of each of two competitor DNAoligonucleotides (5′-TAGACCTGTTAGTGTACATTTGAATTGAAGC-3′; SEQ ID NO:554and 5′-TAGACCTGTTAGGTTGGATTTGTGGCATCA-3′; SEQ ID NO:552), 50 μg yeastRNA (Sigma), and 0.3 nmol of biotin-labeled telomerase-specificoligonucleotide(5′-biotin-TAGACCTGTTA-(rmeG)₂-(rmeU)₄-(rmeG)₄-(rmeU)₄-rmeG-3′; SEQ IDNO:556), were added per ml of the pool. The 2-O-methyribonucleotides ofthe telomerase specific oligonucleotides were complementary to thetelomerase RNA; template region; the deoxyribonucleotides were notcomplementary. The inclusion of competitor, non-specific DNAoligonucleotides increased the efficiency of the purification, as theeffects of nucleic acid binding proteins and other components in themixture that would either bind to the affinity oligonucleotide or removethe telomerase from the mixture were minimized.

[0679] This material was then added to Ultralink immobilized neutravidinplus (Pierce) column material, at a volume of 60 μl of suspension per mlof pool. The column material was pre-blocked twice for 15 minutes eachblocking, with a preparation of WB containing 0.01% Nonidet P-40, 0.5 mgBSA, 0.5 mg/ml lysozyme, 0.05 mg/ml glycogen, and 0.1 mg/ml yeast RNA.The blocking was conducted at 4° C., using a rotating wheel to block thecolumn material thoroughly. After the first blocking step, and beforethe second blocking step, the column material was centrifuged at 200×gfor 2 minutes to pellet the matrix.

[0680] The pool-column mixture was incubated for 8 minutes at 30° C.,and then for an additional 2 hours at 4E° C., on a rotating wheel(approximately 10 rpm; Labindustries) to allow binding. The pool-columnmixture was then centrifuged 200×g for 2 minutes, and the supernatantcontaining unbound material was removed. The pool-column mixture wasthen washed. This washing process included the steps of rinsing thepool-column mixture with WB at 4° C., washing the mixture for 15 minuteswith WB at 4° C., rinsing with WB, washing for 5 minutes at 30° C., withWB containing 0.6 M KGlu, and no Nonidet P-40, washing 5 minutes at 25°C. with WB, and finally, rinsing again with WB. The volume remainingafter the final wash was kept small, in order to yield a ratio of bufferto column material of approximately 1:1.

[0681] Telomerase was eluted from the column material by adding 1 nmolof displacement deoxyoligonucleotide (5′-CA4C₄A4C₂TA₂CAG₂TCTA-3′; SEQ IDNO:557), per ml of column material and incubating at 25° C. for 30minutes. The material was centrifuged for 2 minutes at 14,000 rpm in amicrocentrifuge (Eppendorf), and the eluate collected. The elutionprocedure was repeated twice more, using fresh displacementoligonucleotide each time. As mentioned above, because the displacementoligonucleotide was complementary to the affinity oligonucleotide, itformed a more thermodynamically stable complex with the affinityoligonucleotide than P-40. Thus, addition of the displacementoligonucleotide to an affinity-bound telomerase resulted in efficientelution of telomerase under native conditions. The telomerase appearedto be approximately 50% pure at this stage, as judged by analysis on aprotein gel. The affinity purification of telomerase and elution with adisplacement oligonucleotide is shown in FIG. 26 (panels A and B,respectively). In this Figure, the 2′-O-methyl sugars of the affinityoligonucleotide are indicated by the bold line. The black and shadedoval shapes in this figure are intended to represent graphically theprotein subunits of the present invention.

[0682] The protein concentrations of the extract and material obtainedfollowing Affi-Gel-heparin column chromatography were determined usingthe method of Bradford (Bradford, Anal. Biochem., 72:248 [1976]), usingBSA as the standard. Only a fraction of the telomerase preparation wasfurther purified on a glycerol gradient.

[0683] The sedimentation coefficient of telomerase was determined byglycerol gradient centrifugation, as described in Part I.

[0684] Table 5 below is a purification table for telomerase purifiedaccording to the methods of this Example. The telomerase was enriched12-fold in nuclear extracts, as compared to whole cell extracts, with arecovery of 80%; 85% of telomerase was solubilized from nuclei uponextraction. TABLE 5 Purification of Telomerase Telomerase Telomerase/Purifica- Protein (pmol of Protein/pmol Recovery tion Fraction (mg) RNP)of RNP/mg (%) Factor Nuclear 2020 1720 0.9 100 1 Extract Heparin 1251040 8.3 60 10 Affinity 0.3** 680 2270 40 2670 Glycerol NA* NA* NA* 25NA* Gradient

[0685] E. Telomerase Activity

[0686] At each step in the purification of telomerase, the preparationwas analyzed by three separate assays, one of which was activity, asdescribed in this Example. In general, telomerase assays were done in 40μl containing 0.003-0.3 μl of nuclear extract, 50 mM Tris-Cl (pH 7.5),50 mM KGlu, 10 mM MgCl₂, 1 mM DTT, 125 μM dTTP, 125 μM dGTP, andapproximately 0.2 pmoles of 5′-³²P-labelled oligonucleotide substrate(i.e., approximately 400,000 cpm). Oligonucleotide primers wereheat-denatured prior to their addition to the reaction mixture.Reactions were assembled on ice and incubated for 30 minutes at 25EC.The reactions were stopped by addition of 200 μl of 10 mM Tris-Cl (pH7.5), 15 mM EDTA, 0.6% SDS, and 0.05 mg/ml proteinase K, and incubatedfor at least 30 minutes at 45EC. After ethanol precipitation, theproducts were analyzed on denaturing 8% PAGE gels, as known in the art(See e.g., Sambrook et al., 1989).

[0687] F. Quantitation of Telomerase Activity

[0688] In this Example, quantitation of telomerase activity through thepurification procedure is described. Quantitation was accomplished byassaying the elongation of oligonucleotide primers in the presence ofdGTP and [α-³²P]dTTP. Briefly, 1 μM 5′-(G₄T₄)₂-3′ oligonucleotide wasextended in a 20 μl reaction mixture in the presence of 2 μl of[α-³²P]dTTP (10 mCi/ml, 400 Ci/mmol; 1 Ci=37 GBq), and 125 μM dGTP asdescribed (Lingner et al., Genes Develop., 8:1984 [1994]) and loadedonto an 8% PAGE sequencing gel as described.

[0689] The results of this study are shown in FIG. 28. In lane 1, thereis no telomerase present (i.e., a negative control); lanes 2, 5, 8, and11 contained 0.14 fmol telomerase; lanes 3, 6, 9, and 12 contained 0.42fmol telomerase; and lanes 4, 7, 10, and 13 contained 1.3 fmoltelomerase. Activity was quantitation using a PhosphorImager (MolecularDynamics) using the manufacturer's instructions. It was determined thatunder these conditions, 1 fmol of affinity-purified telomeraseincorporated 21 fmol of dTTP in 30 minutes.

[0690] As shown in FIG. 28, the specific activity of the telomerase didnot change significantly through the purification procedure.Affinity-purified telomerase was fully active. However, it wasdetermined that at high concentrations, an inhibitory activity wasdetected and the activity of crude extracts was not linear. Thus, in theassay shown in FIG. 28, the crude extract was diluted 700-7000-fold.Upon purification, this inhibitory activity was removed and noinhibitory effect was detected in the purified telomerase preparations,even at high enzyme concentrations.

[0691] G. Gel Electrophoresis and Northern Blots

[0692] As stated in Part E, at each step in the purification oftelomerase, the preparation was analyzed by three separate assays. ThisExample describes the gel electrophoresis and blotting procedures usedto quantify telomerase RNA present in fractions and analyze theintegrity of the telomerase ribonucleoprotein particle.

[0693] i) Denaturing Gels and Northern Blots

[0694] In this Example, synthetic T7-transcribed telomerase RNA of knownconcentration served as the standard. Throughout this investigation, theRNA component was used as a measure of telomerase.

[0695] A construct for phage T7 RNA polymerase transcription of E.aediculatus telomerase RNA was produced, using (PCR). The telomerase RNAgene was amplified with primers that annealed to either end of the gene.The primer that annealed at the 5′ end also encoded a hammerheadribozyme sequence to generate the natural 5′ end upon cleavage of thetranscribed RNA, a T7-promoter sequence, and an EcoRI site forsubcloning. The sequence of this 5′ primer was 5′-GCGGGAATTCTAATACGACTCACTATAGGGAAGAAACTCTGATGAGGCCGAAAGGCCGAAACTCCACGAAAGTGGAGTAAGTTTCTCGATAATTGATCTGTAG-3′ (SEQ ID NO:558). The 3′ primerincluded an EarI site for termination of transcription at the natural 3′end, and a BamHI site for cloning. The sequence of this 3′ primer was5′-CGGGGATCCTCTTCAAAAG ATGAGAGGACAGCAAAC-3′ (SEQ ID NO:559). The PCRamplification product was cleaved with EcoRI and BamHI, and subclonedinto the respective sites of pUC 19 (NEB), to give “pEaT7.” Thecorrectness of this insert was confirmed by DNA sequencing. T7transcription was performed as described by Zaug et al., Biochemistry33:14935 [1994], with Earl-linearized plasmid. RNA was gel-purified andthe concentration was determined (an A₂₆₀ of 1=40 μg/ml). This RNA wasused as a standard to determine the telomerase RNA present in variouspreparations of telomerase.

[0696] The signal of hybridization was proportional to the amount oftelomerase RNA, and the derived RNA concentrations were consistent with,but slightly higher than those obtained by native gel electrophoresis.Comparison of the amount of whole telomerase RNA in whole cell RNA toserial dilutions of known T7 RNA transcript concentrations indicatedthat each E. aediculatus cell contained approximately 300,000 telomerasemolecules.

[0697] Visualization of the telomerase was accomplished by Northern blothybridization to its RNA component, using methods as described (Lingeret al., Genes Develop., 8:1984 [1994]). Briefly, RNA (less than or equalto 0.5 μg/lane) was resolved on an 8% PAGE and electroblotted onto aHybond-N membrane (Amersham), as known in the art (see e.g., Sambrook etal., 1989). The blot was hybridized overnight in 10 ml of 4×SSC, 10×Denhardt's solution, 0.1% SDS, and 50 μg/ml denatured herring sperm DNA.After pre-hybridizing for 3 hours, 2×10⁶ cpm probe/ml hybridizationsolution was added. The randomly labelled probe was a PCR-product thatcovered the entire telomerase RNA gene. The blot was washed with severalbuffer changes for 30 minutes in 2×SSC, 0.1% SDS, and then washed for 1hour in 0.1×SSC and 0.1% SDS at 45° C.

[0698] ii) Native Gels and Northern Blots

[0699] In this experiment, the purified telomerase preparation was runon native (i.e., non-denaturing) gels of 3.5% polyacrylamide and 0.33%agarose, as known in the art and described (Lamond and Sproat, [1994],supra). The telomerase comigrated approximately with the xylene cyanoldye.

[0700] The native gel results indicated that telomerase was maintainedas an RNP throughout the purification protocol. FIG. 27 is a photographof a Northern blot showing the mobility of the telomerase in differentfractions on a non-denaturing gel as well as in vitro transcribedtelomerase. In this figure, lane 1 contained 1.5 fmol telomerase RNA,lane 2 contained 4.6 fmol telomerase RNA, lane 3 contained 14 fmoltelomerase RNA, lane 4 contained 41 fmol telomerase RNA, lane 5contained nuclear extract (42 fmol telomerase), lane 6 containedAffi-Gel-heparin-purified telomerase (47 fmol telomerase), lane 7contained affinity-purified telomerase (68 fmol), and lane 8 containedglycerol gradient-purified telomerase (35 fmol).

[0701] As shown in FIG. 27, in nuclear extracts, the telomerase wasassembled into an RNP particle that migrated slower than unassembledtelomerase RNA. Less than 1% free RNA was detected by this method.However, a slower migrating telomerase RNP complex was also sometimesdetected in extracts. Upon purification on the Affi-Gel-heparin column,the telomerase RNP particle did not change in mobility (FIG. 27, lane6). However, upon affinity purification the mobility of the RNA particleslightly increased (FIG. 27, lane 7), perhaps indicating that a proteinsubunit or fragment had been lost. On glycerol gradients, theaffinity-purified telomerase did not change in size, but approximately2% free telomerase RNA was detectable (FIG. 27, lane 8), suggesting thata small amount of disassembly of the RNP particle had occurred.

[0702] H. Telomerase Protein Composition

[0703] In this Example, the analysis of the purified telomerase proteincomposition are described.

[0704] Glycerol gradient fractions obtained as described in Part D, wereseparated on a 4-20% polyacrylamide gel (Novex). Followingelectrophoresis, the gel was stained with Coomassie brilliant blue. FIG.29 shows a photograph of the gel. Lanes 1 and 2 contained molecular massmarkers (Pharmacia) as indicated on the left side of the gel shown inFIG. 29. Lanes 3-5 contained glycerol gradient fraction pools asindicated on the top of the gel (i.e., lane 3 contained fractions 9-14,lane 4 contained fractions 15-22, and lane 5 contained fractions 23-32).Lane 4 contained the pool with 1 pmol of telomerase RNA. In lanes 6-9BSA standards were run at concentrations indicated at the top of the gelin FIG. 29 (i.e., lane 6 contained 0.5 pmol BSA, lane 7 contained 1.5pmol BSA, lane 8 contained 4.5 BSA, and lane 9 contained 15 pmol BSA).

[0705] As shown in FIG. 29, polypeptides with molecular masses of 120and 43 kDa co-purified with the telomerase. The 43 kDa polypeptide wasobserved as a doublet. It was noted that the polypeptide ofapproximately 43 kDa in lane 3 migrated differently than the doublet inlane 4; it may be an unrelated protein. The 120 kDa and 43 kDa doubleteach stained with Coomassie brilliant blue at approximately the level of1 pmol, when compared with BSA standards. Because this fractioncontained 1 pmol of telomerase RNA, all of which was assembled into anRNP particle (See, FIG. 27, lane 8), there appear to be two polypeptidesubunits that are stoichiometric with the telomerase RNA. However, it isalso possible that the two proteins around 43 kDa are separate enzymesubunits.

[0706] Affinity-purified telomerase that was not subjected tofractionation on a glycerol gradient contained additional polypeptideswith apparent molecular masses of 35 and 37 kDa, respectively. Thislatter fraction was estimated to be at least 50% pure. However, the 35kDa and 37 kDa polypeptides that were present in the affinity-purifiedmaterial were not reproducibly separated by glycerol gradientcentrifugation. These polypeptides may be contaminants, as they were notvisible in all activity-containing preparations.

[0707] I. Sedimentation Coefficient

[0708] The sedimentation coefficient for telomerase was determined byglycerol gradient centrifugation. In this Example, nuclear extract andaffinity-purified telomerase were fractionated on 15-40% glycerolgradients containing 20 mM Tris-acetate, with 1 mM MgCl₂, 0.1 mM EDTA,300 mM KGlu, and 1 mM DTT, at pH 7.5. Glycerol gradients were poured in5 ml (13×51 mm) tubes, and centrifuged using an SW55Ti rotor (Beckman)at 55,000 rpm for 14 hours at 4° C.

[0709] Marker proteins were run in a parallel gradient and had asedimentation coefficient of 7.6 S for alcohol dehydrogenase (ADH), 113S for catalase, 17.3 S for apoferritin, and 19.3 S for thyroglobulin.The telomerase peak was identified by native gel electrophoresis ofgradient fractions followed by blot hybridization to its RNA component.

[0710]FIG. 30 is a graph showing the sedimentation coefficient fortelomerase. As shown in this Figure, affinity-purified telomeraseco-sedimented with catalase at 11.5 S, while telomerase in nuclearextracts sedimented slightly faster, peaking around 12.5 S. Therefore,consistent with the mobility of the enzyme in native gels, purifiedtelomerase appears to have lost a proteolytic fragment or a looselyassociated subunit.

[0711] The calculated molecular mass for telomerase, if it is assumed toconsist of one 120 kDa protein subunit, one 43 kDa subunit, and one RNAsubunit of 66 kDa, adds up to a total of 229 kDa. This is in closeagreement with the 232 kDa molecular mass of catalase. However, thesedimentation coefficient is a function of the molecular mass, as wellas the partial specific volume and the frictional coefficient of themolecule, both of which are unknown for the Euplotes telomerase RNP.

[0712] J. Substrate Utilization

[0713] In this Example, the substrate requirements of Euplotestelomerase were investigated. One simple model for DNA end replicationpredicts that after semi-conservative DNA replication, telomeraseextends double-stranded, blunt-ended DNA molecules. In a variation ofthis model, a single-stranded 3′ end is created by a helicase ornuclease after replication. This 3′ end is then used by telomerase forbinding and extension.

[0714] To determine whether telomerase is capable of elongatingblunt-ended molecules, model hairpins were synthesized with telomericrepeats positioned at their 3′ ends. These primer substrates weregel-purified, 5′-end labelled with polynucleotide kinase, heated at 0.4μM to 80° C. for 5 minutes, and then slowly cooled to room temperaturein a heating block, to allow renaturation and helix formation of thehairpins. Substrate mobility on a non-denaturing gel indicated that veryefficient hairpin formation was present, as compared to dimerization.

[0715] Assays were performed with unlabelled 125 μM dGTP, 125 μM dTTP,and 0.02 μM 5′-end-labelled primer (5′-³²P-labelled oligonucleotidesubstrate) in 10 μl reaction mixtures that contained 20 mM Tris-acetate,with 10 mM MgCl₂, 50 mM KGlu, and 1 mM DTT, at pH 7.5. These mixtureswere incubated at 25° C. for 30 minutes. Reactions were stopped byadding formamide loading buffer (i.e., TBE, formamide, bromthymol blue,and cyanol, Sambrook, 1989, supra).

[0716] Primers were incubated without telomerase (“−”), with 5.9 fmol ofaffinity-purified telomerase (“+”), or with 17.6 fmol ofaffinity-purified telomerase (“+++”). Affinity-purified telomerase usedin this assay was dialyzed with a membrane having a molecular cut-off of100 kDa, in order to remove the displacement oligonucleotide. Reactionproducts were separated on an 8% PAGE/urea gel containing 36% formamide,to denature the hairpins. The sequences of the primers used in thisstudy, as well as their lane assignments are shown in Table 6. TABLE 6Primer Sequences Lane Primer Sequence (5′ to 3′) SEQ ID NO: 1-3C₄(A₄C₄)₃CACA(G₄T₄)₃G₄ 560 4-6 C₂(A₄C₄)₃CACA(G₄T₄)₃G₄ 561 7-9(A₄C₄)₃CACA(G₄T₄)₃G₄ 562 10-12 A₂C₄(A₄C₄)₂CACA(G₄T₄)₃G₄ 563 13-15C₄(A₄C₄)₂CACA(G₄T₄)₃ 564 16-18 (A₄C₄)₃CACA(G₄T₄)₃ 565 19-21A₂C₄(A₄C₄)₂CACA(G₄T₄)₃ 566 22-24 C₄(A₄C₄)₂CACA(G₄T₄)₃ 564 25-27C₂(A₄C₄)₂CACA(G₄T₄)₃ 567 28-30 (A₄C₄)₂CACA(G₄T₄)₃ 568

[0717] The gel results are shown in FIG. 31. Lanes 1-15 containedsubstrates with telomeric repeats ending with four G residues. Lanes16-30 contained substrates with telomeric repeats ending with four Tresidues. The putative alignment on the telomerase RNA template isindicated in FIG. 32. It was assumed that the primer sets anneal at twovery different positions in the template shown in FIG. 32 (i.e., Panel Aand Panel B, respectively). This may have affected their binding and/orelongation rate.

[0718]FIG. 33 shows a lighter exposure of lanes 25-30 in FIG. 31. Thelighter exposure of FIG. 33 was taken to permit visualization of thenucleotides that are added and the positions of pausing in elongatedproducts. Percent of substrate elongated for the third lane in each setwas quantified on a PhosphorImager, as indicated on the bottom of FIG.31.

[0719] The substrate efficiencies for these hairpins were compared withdouble-stranded telomere-like substrates with overhangs of differinglengths. A model substrate that ended with four G residues (see lanes1-15 of FIG. 31) was not elongated when it was blunt ended (see lanes1-3). However, slight extension was observed with an overhang length oftwo bases; elongation became efficient when the overhang was at least 4bases in length. The telomerase acted in a similar manner with adouble-stranded substrate that ended with four T residues, with a 6-baseoverhang required for highly efficient elongation. In FIG. 31, the faintbands below the primers in lanes 10-15 that are independent oftelomerase represent shorter oligonucleotides in the primerpreparations.

[0720] The lighter exposure of lanes 25-30 in FIG. 33 shows a ladder ofelongated products, with the darkest bands correlating with the putative5′ boundary of the template (as described by Lingner et al., GenesDevelop., 8:1984 [1994]). The abundance of products that correspond toother positions in the template suggested that pausing and/ordissociation occurs at sites other than the site of translocation withthe purified telomerase.

[0721] As shown in FIG. 31, double-stranded, blunt-endedoligonucleotides were not substrates for telomerase. To determinewhether these molecules would bind to telomerase, a competitionexperiment was performed. In this experiment, 2 nM of 5′-end labeledsubstrate with the sequence (G₄T₄)₂ (SEQ ID NO:114), or a hairpinsubstrate with a six base overhang were extended with 0.125 nMtelomerase (FIG. 31, lanes 25-27). Although the same unlabeledoligonucleotide substrates competed efficiently with labeled substratefor extension, no reduction of activity was observed when thedouble-stranded blunt-ended hairpin oligonucleotides were used ascompetitors, even in the presence of 100-fold excess hairpins.

[0722] These results indicated that double-stranded, blunt-endedoligonucleotides cannot bind to telomerase at the concentrations andconditions tested in this Example. Rather, a single-stranded 3′ end isrequired for binding. It is likely that this 3′ end is required to basepair with the telomerase RNA template.

[0723] K Cloning & Sequencing of the 123 kDa Polypeptide

[0724] In this Example, the cloning of the 123 kDa polypeptide ofEuplotes telomerase (i.e., the 123 kDa protein subunit) is described. Inthis study, an internal fragment of the telomerase gene was amplified byPCR, with oligonucleotide primers designed to match peptide sequencesthat were obtained from the purified polypeptide obtained in Part D,above. The polypeptide sequence was determined using the nanoES tandemmass spectroscopy methods known in the art and described by Calvio etal., RNA 1:724-733 [1995]. The oligonucleotide primers used in thisExample had the following sequences, with positions that were degenerateshown in parentheses— 5′-TCT(G/A)AA(G/A)TA(G/A)TG(T/G/A) (SEQ ID NO:569)GT(G/A/T/C)A(T/G/A)(G/A)TT(G/A) TTCAT-3′, and5′-GCGGATCCATGAA(T/C)CC(A/T)GA (SEQ ID NO:570)(G/A)AA(T/C)CC(A/T)AA(T/C)GT-3′.

[0725] A 50 μl reaction contained 0.2 mM dNTPs, 0.15 μg E. aediculatuschromosomal DNA, 0.5 μl Taq (Boehringer-Mannheim), 0.8 μg of eachprimer, and 1× reaction buffer (Boehringer-Mannheim). The reaction wasincubated in a thermocycler (Perkin-Elmer), using the following—5minutes at 95° C., followed by 30 cycles of 1 minute at 94° C., 1 minuteat 52° C., and 2 minutes at 72° C. The reaction was completed by a 10minute incubation at 72EC.

[0726] A genomic DNA library was prepared from the chromosomal E.aediculatus DNA by cloning blunt-ended DNA into the SmaI site ofpCR-Script plasmid vector FIG. 14 (Stratagene). This library wasscreened by colony hybridization, with the radiolabelled, gel-purifiedPCR product. Plasmid DNA of positive clones was prepared and sequencedby the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci., 74:5463[1977]) or manually, through use of an automated sequencer (ABI). TheDNA sequence of the gene encoding this polypeptide is shown in FIG. 13.The start codon in this sequence inferred from the DNA sequence, islocated at nucleotide position 101, and the open reading frame ends atposition 3193. The genetic code of Euplotes differs from other organismsin that the “UGA” codon encodes a cysteine residue. The amino acidsequence of the polypeptide inferred from the DNA sequence is shown inFIG. 14, and assumes that no unusual amino acids are inserted duringtranslation and no post-translational modification occurs.

[0727] L. Cloning & Sequencing of the 43 kDa Polypeptide

[0728] In this Example, the cloning of the 43 kDa polypeptide oftelomerase (i.e., the 43 kDa protein subunit) is described. In thisstudy, an internal fragment of the corresponding telomerase gene wasamplified by PCR, with oligonucleotide primers designed to match peptidesequences that were obtained from the purified polypeptide obtained inPart D, above. The polypeptide sequence was determined using the nanoEStandem mass spectroscopy methods known in the art and described byCalvio et al., supra. The oligonucleotide primers used in this Examplehad the following sequences--5′-NNNGTNAC(C/T/A)GG(C/T/A)AT(C/T/A)AA(C/T)AA-3′ (SEQ ID NO:571), and5′-(T/G/A)GC (T/G/A)GT(C/T)TC(T/C)TG(G/A)TC(G/A)TT(G/A)TA-3′ (SEQ IDNO:572). In this sequence, “N” indicates the presence of any of the fournucleotides (i.e., A, T, G, or C).

[0729] The PCR was performed as described in Part K.

[0730] A genomic DNA library was prepared and screened as described inPart K. The DNA sequence of the gene encoding this polypeptide is shownin FIG. 34. Three potential reading frames are shown for this sequence,as shown in FIG. 35. For clarity, the amino acid sequence is indicatedbelow the nucleotide sequence in all three reading frames. These readingframes are designated as “a,” “b,” and “c”. A possible start codon isencoded at nucleotide position 84 in reading frame “c.” The codingregion could end at position 1501 in reading frame “b.” Early stopcodons, indicated by asterisks in this figure, occur in all threereading frames between nucleotide position 337-350.

[0731] Further downstream, the protein sequence appears to be encoded bydifferent reading frames, as none of the three frames is uninterruptedby stop codons. Furthermore, peptide sequences from purified protein areencoded in all three frames. Therefore, this gene appears to containintervening sequences, or in the alternative, the RNA is edited. Otherpossibilities include ribosomal frame-shifting or sequence errors.However, the homology to the La-protein sequence remains of significantinterest. Again, in Euplotes, the “UGA” codon encodes a cysteineresidue.

[0732] M. Amino Acid and Nucleic Acid Comparisons

[0733] In this Example, comparisons between various reported sequencesand the sequences of the 123 kDa and 43 kDa telomerase subunitpolypeptides were made.

[0734] i) Comparisons with the 123 kDa E. aediculatus Telomerase Subunit

[0735] The amino acid sequence of the 123 kDa Euplotes aediculatuspolypeptide was compared with the sequence of the 80 kDa telomeraseprotein subunit of Tetrahymena thermophila (GenBank accession #U25641)to investigate their similarity. The nucleotide sequence as obtainedfrom GenBank encoding this protein is shown in FIG. 42. The amino acidsequence of this protein as obtained from GenBank is shown in FIG. 43.The sequence comparison between the 123 kDa E. aediculatus and 80 kDa T.thermophila is shown in FIG. 36. In this figure, the E. aediculatussequence is the upper sequence, while the T. thermophila sequence is thelower sequence. The observed identity was determined to be approximately19%, while the percent similarity was approximately 45%, values similarto what would be observed with any random protein sequence. In FIGS.36-39, identities are indicated by vertical bars, while single dotsbetween the sequences indicate somewhat similar amino acids, and doubledots between the sequences indicate more similar amino acids.

[0736] The amino acid sequence of the 123 kDa Euplotes aediculatuspolypeptide was also compared with the sequence of the 95 kDa telomeraseprotein subunit of Tetrahymena thermophila (GenBank accession #U25642),to investigate their similarity. The nucleotide sequence as obtainedfrom GenBank encoding this protein is shown in FIG. 44. The amino acidsequence of this protein as obtained from GenBank is shown in FIG. 45.This sequence comparison is shown in FIG. 37. In this figure, the E.aediculatus sequence is the upper sequence), while the T. thermophilasequence is the lower sequence. The observed identity was determined tobe approximately 20%, while the percent similarity was approximately43%, values similar to what would be observed with any random proteinsequence.

[0737] Significantly, the amino acid sequence of the 123 kDa E.aediculatus polypeptide contains the five motifs characteristic ofreverse transcriptases. The 123 kDa polypeptide was also compared withthe polymerase domains of various reverse transcriptases. FIG. 40 showsthe alignment of the 123 kDa polypeptide with the putative yeast homolog(L8543.12 or ESTp). The amino acid sequence of L8543.12 obtained fromGenBank is shown in FIG. 46.

[0738] Four motifs (A, B, C, and D) were included in this comparison. Inthis FIG. 40, highly conserved residues are indicated by white letterson a black background. Residues of the E. aediculatus sequences that areconserved in the other sequence are indicated in bold; the “h” indicatesthe presence of a hydrophobic amino acid. The numerals located betweenamino acid residues of the motifs indicates the length of gaps in thesequences. For example, the “100” shown between motifs A and B reflectsa 100 amino acid gap in the sequence between the motifs.

[0739] As noted above, Genbank searches identified a yeast protein(Genbank accession #u20618), and gene L8543.12 (Est2) containing orencoding amino acid sequence that shows some homology to the E.aediculatus 123 kDa telomerase subunit. Based on the observations thatboth proteins contain reverse transcriptase motifs in their C-terminalregions; both proteins share similarity in regions outside the reversetranscriptase motif; the proteins are similarly basic (pI=10.1 for E.aediculatus and pI=10.0 for the yeast); and both proteins are large (123kDa for E. aediculatus and 103 kDa for the yeast), these sequencescomprise the catalytic core of their respective telomerases. It wascontemplated based on this observation of homology in twophylogenetically distinct organisms as E. aediculatus and yeast, thathuman telomerase would contain a protein that has the samecharacteristics (i.e., reverse transcriptase motifs, is basic, and large[>100 kDa]).

[0740] ii) Comparisons with the 43 kDa E. aediculatus Telomerase Subunit

[0741] The amino acid sequence of the “La-domain” of the 43 kDa Euplotesaediculatus polypeptide was compared with the sequence of the 95 kDatelomerase protein subunit of Tetrahymena thermophila (described above)to investigate their similarity. This sequence comparison is shown inFIG. 38, while the T. thermophila sequence is the lower sequence. Theobserved identity was determined to be approximately 23%, while thepercent similarity was approximately 46%, values similar to what wouldbe observed with any random protein sequence.

[0742] The amino acid sequence of the “La-domain” of the 43 kDa Euplotesaediculatus polypeptide was compared with the sequence of the 80 kDatelomerase protein subunit of Tetrahymena thermophila (described above)to investigate their similarity. This sequence comparison is shown inFIG. 39. In this figure, the E. aediculatus sequence is the uppersequence, while the T. thermophila sequence is the lower sequence. Theobserved identity was determined to be approximately 26%, while thepercent similarity was approximately 49%, values similar to what wouldbe observed with any random protein sequence.

[0743] The amino acid sequence of a domain of the 43 kDa E. aediculatuspolypeptide was also compared with La proteins from various otherorganisms. These comparisons are shown in FIG. 41. In this Figure,highly conserved residues are indicated by white letters on a blackbackground. Residues of the E. aediculatus sequences that are conservedin the other sequence are indicated in bold.

[0744] N. Identification of Telomerase Protein Subunits in AnotherOrganism

[0745] In this Example, the sequences identified in the previousExamples above were used to identify the telomerase protein subunits ofOxytricha trifallax, a ciliate that is very distantly related to E.aediculatus. Primers were chosen based on the conserved region of the E.aediculatus 123 kDa polypeptide which comprised the reversetranscriptase domain motifs. Suitable primers were synthesized and usedin a PCR reaction with total DNA from Oxytricha. The Oxytricha DNA wasprepared according to methods known in the art. The PCR products werethen cloned and sequenced using methods known in the art.

[0746] The oligonucleotide sequences used as the primers were asfollows: 5′-(T/C)A(A/G)AC(T/A/C)AA(G/A)GG (SEQ ID NO:573)(T/A/C)AT(T/C)CC(C/T/A)(C/T)A(G/A) GG-3′ and5′-(G/A/T)GT(G/A/T)ATNA(G/A)NA (SEQ ID NO:574)(G/A)(G/A)TA(G/A)TC(G/A)TC-3′.

[0747] Positions that were degenerate are shown in parentheses, with thealternative bases shown within the parenthesis. “N” represents any ofthe four nucleotides.

[0748] In the PCR reaction, a 50 μl reaction contained 0.2 mM dNTPs, 0.3μg Oxytricha trifallax chromosomal DNA, 1 μl Taq polymerase(Boehringer-Mannheim), 2 micromolar of each primer, 1× reaction buffer(Boehringer-Mannheim). The reaction was incubated in a thermocycler(Perkin-Elmer) under the following conditions: 5 min at 95° C., 30cycles consisting of 1 min at 94° C., 1 min at 53° C., and 1 min at 72°C., followed by a 10 min incubation at 72° C. The PCR-product wasgel-purified and sequenced by the dideoxy-method (e.g., Sanger et al.,Proc. Natl. Acad. Sci. 74, 5463-5467 (1977).

[0749] The deduced amino acid sequence of the PCR product was determinedand compared with the E. aediculatus sequence. FIG. 47 shows thealignment of these sequences, with the O. trifallax sequence shown inthe top row, and the E. aediculatus sequence shown in the bottom row. Ascan be seen from this figure, there is a great deal of homology betweenthe O. trifallax polypeptide sequence identified in this Example withthe E. aediculatus polypeptide sequence. Thus, it is clear that thesequences identified in the present invention are useful for theidentification of homologous telomerase protein subunits in othereukaryotic organisms. Indeed, development of the present invention hasidentified homologous telomerase sequences in multiple, diverse species,as described herein.

[0750] O. Identification of Tetrahymena Telomerase Sequences

[0751] In this Example, a Tetrahymena clone was produced that shareshomology with the Euplotes sequences, and EST2p.

[0752] This experiment utilized PCR with degenerate oligonucleotideprimers directed against conserved motifs to identify regions ofhomology between Tetrahymena, Euplotes, and EST2p sequences. The PCRmethod used in this Example is a novel method designed to amplifyspecifically rare DNA sequences from complex mixtures. This methodavoids the problem of amplification of DNA products with the same PCRprimer at both ends (i.e., single primer products) commonly encounteredin PCR cloning methods. These single primer products produce unwantedbackground and can often obscure the amplification and detection of thedesired two-primer product. The method used in this experimentpreferentially selects for two-primer products. In particular, oneprimer is biotinylated and the other is not. After several rounds of PCRamplification, the products are purified using streptavidin magneticbeads and two primer products are specifically eluted using heatdenaturation. This method finds use in settings other than theexperiments described in this Example. Indeed, this method finds use inapplication in which it is desired to specifically amplify rare DNAsequences, including the preliminary steps in cloning methods such as 5′and 3′ RACE, and any method that uses degenerate primers in PCR.

[0753] A first PCR run was conducted using Tetrahymena templatemacronuclear DNA isolated using methods known in the art, and the 24-merforward primer with the sequence 5′biotin-GCCTATTT(TC)TT(TC)TA(TC)(GATC)(GATC) (GATC)AC(GATC)GA-3′ (SEQ IDNO:575) designated as “K231,” corresponding to the FFYXTE SEQ ID NO:360region, and the 23-mer reverse primer with the sequence5′-CCAGATAT(GATC)A (TGA)(GATC)A(AG)(AG)AA(AG)TC(AG)TC-3′ (SEQ IDNO:576), designated as “K220,” corresponding to the DDFL(FIL)I (SEQ IDNO:577) region. This PCR reaction contained 2.5 μl DNA (50 ng), 4 μl ofeach primer (20 μM), 3 μl 10×PCR buffer, 3 μl 10×dNTPs, 2 μl Mg, 0.3 μlTaq, and 11.2 μl dH₂O. The mixture was cycled for 8 cycles of 94° C. for45 seconds, 37° C. for 45 seconds, and 72° C. for 1 minute.

[0754] This PCR reaction was bound to 200 μl streptavidin magneticbeads, washed with 200 μl TE, resuspended in 20 μl dH₂O and thenheat-denatured by boiling at 100° C. for 2 minutes. The beads werepulled down and the eluate removed. Then, 2.5 μl of this eluate wassubsequently reamplified using the above conditions, with the exceptionbeing that 0.3 μl of α-³²P dATP was included, and the PCR was carriedout for 33 cycles. This reaction was run a 5% denaturing polyacrylamidegel, and the appropriate region was cut out of the gel. These productswere then reamplified for an additional 34 cycles, under the conditionslisted above, with the exception being that a 42° C. annealingtemperature was used.

[0755] A second PCR run was conducted using Tetrahymena macronuclear DNAtemplate isolated using methods known in the art, and the 23-mer forwardprimer with the sequence 5′-ACAATG(CA)G(GATC)(TCA)T(GATC)(TCA)T(GATC)CC(GATC)AA(AG)AA-3′ (SEQ ID NO:578), designated as “K228,” correspondingto the region R(LI)(LI)PKK (SEQ ID NO:579), and a reverse primer withthe sequence 5′-ACGAATC(GT)(GATC)G(TAG)AT(GATC)(GC)(TA)(AG)TC(AG)TA(AG)CA 3′ (SEQ ID NO:580), designated“K224,” corresponding to the CYDSIPR (SEQ ID NO:581) region. This PCRreaction contained 2.5 μl DNA (50 ng), 4 μl of each primer (20 μM), 3 μl10×PCR buffer, 3 μl 10×dNTPs, 2 μl Mg, 0.3 μl α-³²P dATP, 0.3 μl Taq,and 10.9 μl dH₂O. This reaction was run on a 5% denaturingpolyacrylamide gel, and the appropriate region was cut out of the gel.These products were reamplified for an additional 34 cycles, under theconditions listed above, with the exception being that a 42° C.annealing temperature was used.

[0756] Ten μl of the reaction product from run 1 were bound tostreptavidin-coated magnetic beads in 200 μl TE. The beads were washedwith 200 μl TE, and then resuspended in 20 μl of dH₂O, heat denatured,and the eluate was removed. The reaction product from run 2 was thenadded to the beads and diluted with 30 μl 0.5×SSC. The mixture washeated from 94° C. to 50° C. The eluate was removed and the beads werewashed three times in 0.5×SSC at 55EC. The beads were then resuspendedin 20 μl dH₂O, heat denatured, and the eluate was removed, designated as“round 1 eluate” and saved.

[0757] To isolate the Tetrahymena band, the round 1 eluate wasreamplified with the forward primer K228 and reverse primer K227 withthe sequence 5′-CAATTCTC(AG)TA(AG)CA(GATC)(CG)(TA)(CT)TT(AGT)AT(GA)TC-3′(SEQ ID NO:582), corresponding to the DIKSCYD (SEQ ID NO:583) region.The PCR reactions were conducted as described above. The reactionproducts were run on a 5% polyacrylamide gel; the band corresponding toapproximately 295 nucleotides was cut from the gel and sequenced.

[0758] The clone designated as 168-3 was sequenced. The DNA sequence(including the primer sequences) was found to be:GATTACTCCCGAAGAAAGGATCTTTCCGTCCAA (SEQ ID NO:584)TCATGACTTTCTTAAGAAAGGACAAGCAAAAAA ATATTAAGTTAAATCTAAATTAAATTCTAATGGATAGCCAACTTGTGTTTAGGAATTTAAAAGACA TGCTGGGATAAAAGATAGGATACTCAGTCTTTGATAATAAACAAATTTCAGAAAAATTTGCCTAAT TCATAGAGAAATGGAAAAATAAAGGAAGACCTCAGCTATATTATGTCACTCTAGACATAAAGACTT GCTAC.

[0759] Additional sequence of this gene was obtained by PCR using oneunique primer designed to match the sequence from 168-3 (“K297” with thesequence 5′-GAGTGACATAATATACGTGA-3′ (SEQ ID NO:585); and the K231(FFYXTE; SEQ ID NO:360) primer. The sequence of the fragment obtainedfrom this reaction, together with 168-3 is as follows (without theprimer sequences): AAACACAAGGAAGGAAGTCAAATATTCTATTAC (SEQ ID NO:586)CGTAAACCAATATGGAAATTAGTGAGTAAATTA ACTATTGTCAAAGTAAGAATTTAGTTTTCTGAAAAGAATAAATAAATGAAAAATAATTTTTATCAA AAAATTTAGCTTGAAGAGGAGAATTTGGAAAAAGTTGAAGAAAAATTGATACCAGAAGATTCATTT TAGAAATACCCTCAAGGAAAGCTAAGGATTATACCTAAAAAAGGATCTTTCCGTCCAATCATGACT TTCTTAAGAAAGGACAAGCAAAAAAATATTAAGTTAAATCTAAATTAAATTCTAATGGATAGCCAA CTTGTGTTTAGGAATTTAAAAGACATGCTGGGATAAAAGATAGGATACTCAGTCTTTGATAATAAA CAAATTTCAGAAAAATTTGCCTAATTCATAGAGAAATGGAAAAATAAAGGAAGACCTCAGCTATAT TATGTCACTCTA.

[0760] The amino acid sequence corresponding to this DNA fragment wasfound to be: KHKEGSQIFYYRKPIWKLVSKLTIVKVRIQFSE (SEQ ID NO:228)KNKQMKNNFYQKIQLEEENLEKVEEKLIPEDSF QKYPQGKLRIIPKKGSFRPIMTFLRKDKQKNIKLNLNQILMDSQLVFRNLKDMLGQKIGYSVFDNK QISEKFAQFIEKWKNKGRPQLYYVTL.

[0761] This amino acid sequence was then aligned with other telomerasegenes (EST2p, and Euplotes). The alignment is shown in FIG. 53. Aconsensus sequence is also shown in this Figure.

[0762] P. Identification of Schizosaccharomyces pombe TelomeraseSequences

[0763] In this Example, the tez1 sequence of S. pombe was identified asa homolog of the E. aediculatus p123, and S. cerevisiae Est2p.

[0764]FIG. 55 provides an overall summary of these experiments. In thisFigure, the top portion (Panel A) shows the relationship of twooverlapping genomic clones, and the 5825 bp portion that was sequenced.The region designated at “tez1⁺” is the protein coding region, with theflanking sequences indicated as well, the box underneath the 5825 bpregion is an approximately 2 kb HindIII fragment that was used to makethe tez1 disruption construct, as described below.

[0765] The bottom half of FIG. 55 (Panel B) is a “close-up” schematic ofthis same region of DNA. The sequence designated as “original PCR” isthe original degenerate PCR fragment that was generated with adegenerate oligonucleotide primer pair designed based on Euplotessequence motif 4 (B′) and motif 5 (C), as described.

[0766] i) PCR with Degenerate Primers

[0767] PCR using degenerate primers was used to find the homolog of theE. aediculatus p123 in S. pombe. FIG. 56 shows the sequences of thedegenerate primers (designated as “poly 4” and “poly 1”) used in thisreaction. The PCR runs were conducted using the same buffer as describedin previous Examples (See e.g., Part K, above), with a 5 minute ramptime at 94° C., followed by 30 cycles of 94° C. for 30 seconds, 50° C.for 45 seconds, and 72° C. for 30 seconds, and 7 minutes at 72° C.,followed by storage at 4° C. PCR runs were conducted using variedconditions, (i.e., various concentrations of S. pombe DNA and MgCl₂concentrations). The PCR products were run on agarose gels and stainedwith ethidium bromide as described above. Several PCR runs resulted inthe production of three bands (designated as “T,” “M,” and “B”). Thesebands were re-amplified and run on gels using the same conditions asdescribed above. Four bands were observed following thisre-amplification (“T,” “M1,” “M2,” and “B”), as shown in FIG. 57. Thesefour bands were then re-amplified using the same conditions as describedabove. The third band from the top of the lane in FIG. 57 was identifiedas containing the correct sequence for a telomerase protein. The PCRproduct designated as M2 was found to show a reasonable match with othertelomerase proteins, as indicated in FIG. 58. In addition to thealignment shown, this Figure also shows the actual sequence of tez1. Inthis Figure, the asterisks indicate residues shared with all foursequences (Oxytricha “Ot”; E. aediculatus “Ea_p123”; S. cerevisiae“Sc_p103”; and M2), while the circles (i.e., dots) indicate similaramino acid residues.

[0768] ii) 3′ RT PCR

[0769] To obtain additional sequence information, 3′ and 5′RT PCR wereconducted on the telomerase candidate identified in FIG. 58. FIG. 59provides a schematic of the 3′ RT PCR strategy used. First, cDNA wasprepared from mRNA using the oligonucleotide primer “Q_(T),” (5′-CCA GTGAGC AGA GTG ACG AGG ACT CGA GCT CAA GCT TTT TTT TTT TTT TT-3′; SEQ IDNO:587), then using this cDNA as a template for PCR with “Q_(O)” (5′-CCAGTG AGC AGA GTG ACG-3′; SEQ ID NO:588), and a primer designed based onthe original degenerated PCR reaction (i.e., “M2-T” with the sequence5′-G TGT CAT TTC TAT ATG GAA GAT TTG ATT GAT G-3′; SEQ ID NO:589). Thesecond PCR reaction (i.e., nested PCR) with “Q_(l)” (5′-GAG GAC TCG AGCTCA AGC-3′; SEQ ID NO:590), and another PCR primer designed withsequence derived from the original degenerate PCR reaction or “M2-T2”(5′-AC CTA TCG TTT ACG AAA AAG AAA GGA TCA GTG-3′; SEQ ID NO:591). Thebuffers used in this PCR were the same as described above, withamplification conducted beginning with a ramp up of 94° for 5 min,followed by 30 cycles of 94° for 30 sec, 55° C. for 30 sec, and 72° C.for 3 min, followed by 7 minutes at 72° C. The reaction products werestored at 4° C. until use.

[0770] iii) Screening of Genomic and cDNA Libraries

[0771] After obtaining this additional sequence information, severalgenomic and cDNA libraries were screened to identify any libraries thatcontain this telomerase candidate gene. The approach used, as well asthe libraries and results are shown in FIG. 60. In this Figure, Panel Alists the libraries tested in this experiment; Panel B shows the regionsused; Panels C and D show the dot blot hybridization results obtainedwith these libraries. Positive libraries were then screened by colonyhybridization to obtain genomic and cDNA version of tez1 gene. In thisexperiment, approximately 3×10⁴ colonies from the HindIII genomiclibrary were screened and six positive clones were identified(approximately 0.01%). DNA was then prepared from two independent clones(A5 and B2). FIG. 61 shows the results obtained with theHindIII-digested A5 and B2 positive genomic clones.

[0772] In addition, cDNA REP libraries were used. Approximately 3×10⁵colonies were screened, and 5 positive clones were identified (0.002%).DNA was prepared from three independent clones (2-3, 4-1, and 5-20). Inlater experiments, it was determined that clones 2-3 and 5-20 containedidentical inserts.

[0773] iv) 5′RT PCR

[0774] As the cDNA version of gene produced to this point was notcomplete, 5′ RT-PCR was conducted to obtain a full length clone. Thestrategy is schematically shown in FIG. 62. In this experiment, cDNA wasprepared using DNA oligonucleotide primer “M2-B” (5′-CAC TGA TCC TTT CTTTTT CGT AAA CGA TAG GT-3′; SEQ ID NO:592) and “M2-B2” (5′-C ATC AAT CAAATC TTC CAT ATA GAA ATG ACA-3′; SEQ ID NO:593), designed from knownregions of tez1 identified previously. An oligonucleotide linker PCRAdapt SfiI with a phosphorylated 5′ end (“P”) (P-GGG CCG TGT TGG CCT AGTTCT CTG CTC-3′ SEQ ID NO:594; was then ligated at the 3′ end of thiscDNA, and this construct was used as the template for nested PCR. In thefirst round of PCR, PCR Adapt SFI and M2-B were used as the primers;while PCR Adapt SfiI (5′-GAG GAG GAG AAG AGC AGA GAA CTA GGC CAA CAC GCCCC-3′; SEQ ID NO:595), and M2-B2 were used as primers in the secondround. Nested PCR was used to increase specificity of reaction.

[0775] v) Sequence Alignments

[0776] Once the sequence of tez1 was identified, it was compared withsequences previously described. FIG. 63 shows the alignment of RTdomains from telomerase catalytic subunits of S. pombe (“S.p. Tez1p”),S. cerevisiae (“S.c. Est2p”), and E. aediculatus p123 (“E.a. p123”). Inthis Figure, “h” indicates hydrophobic residues, while “p” indicatessmall polar residues, and “c” indicates charged residues. The amino acidresidues indicated above the alignment show a known consensus RT motifof Y. Xiong and T. H. Eickbush (Y. Xiong and T. H. Eickbush, EMBO J., 9:3353-3362 [1990]). The asterisks indicate the residues that areconserved for all three proteins. “Motif O” is identified herein and inFIG. 63 as a motif specific to this telomerase subunit and not found inreverse transcriptases in general. It is therefore valuable inidentifying other amino acid sequences as telomerase catalytic subunits.

[0777]FIG. 64 shows the alignment of entire sequences from Euplotes(“Ea_p123”), S. cerevisiae (“Sc_Est2p”), and S. pombe (“Sp_Tez1p”). InPanel A, the shaded areas indicate residues shared between twosequences. In Panel B, the shaded areas indicate residues shared betweenall three sequences.

[0778] vi) Genetic Disruption of Tez1

[0779] In this Example, the effects of disruption of tez1 wereinvestigated. As telomerase is involved in telomere maintenance, it washypothesized that if tez1 were indeed a telomerase component, disruptionof tez1 would cause gradual telomere shortening.

[0780] In these experiments, homologous recombination was used todisrupt the tez1 gene in S. pombe specifically. This approach isschematically illustrated in FIG. 65. As indicated in FIG. 65, wild typetez1 was replaced with a fragment containing the ura4 or LEU2 marker.

[0781] The disruption of tez1 gene was confirmed by PCR (FIG. 66), and aSouthern blot was performed to check for telomere length. FIG. 67 showsthe Southern blot results for this experiment. Because an ApaIrestriction enzyme site is present immediately adjacent to telomericsequence in S. pombe, ApaI digestion of S. pombe genomic DNApreparations permits analysis of telomere length. Thus, DNA from S.pombe was digested with ApaI and the digestion products were run on anagarose gel and probed with a telomeric sequence-specific probe todetermine whether the telomeres of disrupted S. pombe cells wereshortened. The results are shown in FIG. 67. From these results, it wasclear that disruption of the tez1 gene caused a shortening of thetelomeres.

[0782] Q. Cloning and Characterization of Human Telomerase Protein andcDNA

[0783] In this Example, the nucleic and amino acid sequence informationfor human telomerase was determined. Partial homologous sequences werefirst identified in a BLAST search conducted using the Euplotes 123 kDapeptide and nucleic acid sequences, as well as Schizosaccharomycesprotein and corresponding cDNA (tez1) sequences. The human sequences(also referred to as “hTCP 1.1”) were identified from a partial cDNAclone (clone 712562). Sequences from this clone were aligned with thesequences determined as described in previous Examples.

[0784]FIG. 1 shows the sequence alignment of the Euplotes (“p123”),Schizosaccharomyces (“tez1”), Est2p (i.e., the S. cerevisiae proteinencoded by the Est2 nucleic acid sequence, and also referred to hereinas “L8543.12”), and the human homolog identified in this comparisonsearch. FIG. 51 shows the amino acid sequence of tez1, while FIG. 52shows the DNA sequence of tez1. In FIG. 52, the introns and othernon-coding regions, are shown in lower case, while the exons (i.e.,coding regions) are shown in upper case.

[0785] As shown in the Figures, there are regions that are highlyconserved among these proteins. For example, as shown in FIG. 1, thereare regions of identity in “Motif 0,” “Motif 1,” “Motif 2,” and “Motif3.” The identical amino acids are indicated with an asterisk (*), whilethe similar amino acid residues are indicated by a circle (). Thisindicates that there are regions within the telomerase motifs that areconserved among a wide variety of eukaryotes, ranging from yeast tociliates to humans. It is contemplated that additional organisms willlikewise contain such conserved regions of sequence. FIG. 49 shows thepartial amino acid sequence of the human telomerase motifs, while FIG.50 shows the corresponding DNA sequence.

[0786] Sanger dideoxy sequencing and other methods were used, as knownin the art to obtain complete sequence information of clone 712562. Someof the primers used in the sequencing are shown in Table 7. Theseprimers were designed to hybridize to the clone, based on sequencecomplementarity to either plasmid backbone sequence or the sequence ofthe human cDNA insert in the clone. TABLE 7 Primers Primer Sequence SEQID NO: TCP1.1 GTGAAGGCACTGTTCAGCG 377 TCP1.2 GTGGATGATTTCTTGTTGG 381TCP1.3 ATGCTCCTGCGTTTGGTGG 596 TCP1.4 CTGGACACTCAGCCCTTGG 382 TCP1.5GGCAGGTGTGCTGGACACT 383 TCP1.6 TTTGATGATGCTGGCGATG 384 TCP1.7GGGGCTCGTCTTCTACAGG 385 TCP1.8 CAGCAGGAGGATCTTGTAG 386 TCP1.9TGACCCCAGGAGTGGCACG 387 TCP1.10 TCAAGCTGACTCGACACCG 388 TCP1.11CGGCGTGACAGGGCTGC 389 TCP1.12 GCTGAAGGCTGAGTGTCC 390 TCP1.13TAGTCCATGTTCACAATCG 391

[0787] From these experiments, it was determined that the EcoRI-NotIinsert of clone 712562 contains only a partial open reading frame forthe human telomerase protein, although it may encode an active fragmentof that protein. The open reading frame in the clone encodes anapproximately 63 kD protein. The sequence of the longest open readingframe identified is shown in FIG. 68. The ORF begins at the ATG codonwith the “met” indicated in the Figure. The poly A tail at the 3′ end ofthe sequence is also shown. FIG. 69 shows a tentative, preliminaryalignment of telomerase reverse transcriptase proteins from the humansequence (human Telomerase Core Protein 1, “Hs TCP1”), E. aediculatusp123 (“Ep p123”), S. pombe tez1 (“Sp Tez1”), S. cerevisiae EST2 (“ScEst2”), and consensus sequence. In this Figure various motifs areindicated.

[0788] To obtain a full-length clone, probing of a cDNA library and5′-RACE were used to obtain clones encoding portions of the previouslyuncloned regions. In these experiments, RACE (Rapid Amplification ofcDNA Ends; See e.g., M. A. Frohman, “RACE: Rapid Amplification of cDNAEnds,” in Innis et al. (eds), PCR Protocols: A Guide to Methods andApplications [1990], pp. 28-38; and Frohman et al., Proc. Natl. Acad.Sci., 85:8998-9002 [1988]) was used to generate material for sequenceanalysis. Four such clones were generated and used to provide additional5′ sequence information (pFWRP5, 6, 19, and 20).

[0789] In addition, human cDNA libraries (inserted into lambda) wereprobed with the EcoRI-NotI fragment of the clone. One lambda clone,designated “lambda 25-1.1” (ATCC accession #209024), was identified ascontaining complementary sequences. FIG. 75 shows a restriction map ofthis lambda clone. The human cDNA insert from this clone was subclonedas an EcoRI restriction fragment into the EcoRI site of commerciallyavailable phagemid pBluescriptIISK+ (Stratagene), to create the plasmid“pGRN121,” which was deposited with the ATCC (ATCC accession #209016).Preliminary results indicated that plasmid pGRN121 contains the entireopen reading frame (ORF) sequence encoding the human telomerase protein.

[0790] The cDNA insert of plasmid pGRN121 was sequenced using techniquesknown in the art. FIG. 70 provides a restriction site and function mapof plasmid pGRN121 identified based on this preliminary work. Theresults of this preliminary sequence analysis are shown in FIG. 71. Fromthis analysis, and as shown in FIG. 70, a putative start site for thecoding region was identified at approximately 50 nucleotides from theEcoRI site (located at position 707), and the location of thetelomerase-specific motifs, “FFYVTE” (SEQ ID NO:361), “PKP,” “AYD,”“QG”, and “DD,” were identified, in addition to a putative stop site atnucleotide #3571 (See, FIG. 72, which shows the DNA and correspondingamino acid sequences for the open reading frames in the sequence (“a”,“b”, and “c”)). However, due to the preliminary nature of the earlysequencing work, the reading frames for the various motifs were foundnot to be in alignment.

[0791] Additional analysis conducted on the pGRN121 indicated that theplasmid contained significant portions from the 5′-end of the codingsequence not present on clone 712562. Furthermore, pGRN121 was found tocontain a variant coding sequence that includes an insert ofapproximately 182 nucleotides. This insert was found to be absent fromthe clone. As with the E. aediculatus sequences, such variants can betested in functional assays, such as telomerase assays to detect thepresence of functional telomerase in a sample.

[0792] Further sequence analysis resolved the cDNA sequence of pGRN121to provide a contiguous open reading frame that encodes a protein ofmolecular weight of approximately 127,000 daltons, and 1132 amino acidsas shown in FIG. 74. A refined map of pGRN121 based on this analysis, isprovided in FIG. 73. The results of additional sequence analysis of thehTRT cDNA are presented in FIG. 16 SEQ ID NO:1.

Example 2 Correlation of hTRT Abundance and Cell Immortality

[0793] The relative abundance of hTRT mRNA was assessed in sixtelomerase-negative mortal cell strains and six telomerase-positiveimmortal cell lines (FIG. 5). The steady state level of hTRT mRNA wassignificantly increased in immortal cell lines that had previously beenshown to have active telomerase. Lower levels of the hTRT mRNA weredetected in some telomerase-negative cell strains.

[0794] RT-PCR for hTRT, hTR, TP 1 (telomerase-associated protein relatedto Tetrahymena p80 [Harrington et al., 1997, Science 275:973; Nakayamaet al., 1997, Cell 88:875]) and GAPDH (to normalize for equal amounts ofRNA template) was carried out on RNA derived from the following cells:(1) human fetal lung fibroblasts GFL, (2) human fetal skin fibroblastsGFS, (3) adult prostate stromal fibroblasts 31 YO, (4) human fetal kneesynovial fibroblasts HSF, (5) neonatal foreskin fibroblasts BJ, (6)human fetal lung fibroblasts IMR90, and immortalized cell lines: (7)melanoma LOX IMVI, (8) leukemia U251, (9) NCI H23 lung carcinoma, (10)colon adenocarcinoma SW620, (11) breast tumor MCF7, (12) 293 adenovirusE1 transformed human embryonic kidney cell line.

[0795] hTRT nucleic acid was amplified from cDNA using oligonucleotideprimers Lt5 and Lt6 (Table 2) for a total of 31 cycles (94° C. 45 s, 60°C. 45 s, 72° C. 90 s). GAPDH was amplified using primers K136(5′-CTCAGACACCATGGGGAA GGTGA; SEQ ID NO:552) and K137(5′-ATGATCTTGAGGCTGTTGTCATA; SEQ ID NO:553) for a total of 16 cycles(94° C. 45 s, 55° C. 45 s, 72° C. 90 s). hTR was amplified using primersF3b (5′-TCTAACCCTAACTGAGAAGGGCGTAG; SEQ ID NO:597) and R3c(5′-GTTTGCTCTAGAATGAACGGTGGAAG; SEQ ID NO:598) for a total of 22 cycles(94° C. 45 s, 55° C. 45 s, 72° C. 90 s). TP1 mRNA was amplified usingprimers TP1.1 and TP1.2 for 28 cycles (cycles the same as hTRT).Reaction products were resolved on an 8% polyacrylamide gel, stainedwith SYBR Green (Molecular Probes) and visualized by scanning on a Storm860 (Molecular Dynamics). The results, shown in FIG. 5, demonstrate thathTRT mRNA levels correlate directly with telomerase activity levels inthe cells tested.

Example 3 Characterization of an hTRT Intronic Sequence

[0796] A putative intron was first identified by PCR amplification ofhuman genomic DNA, as described in this example, and subsequentlyconfirmed by sequencing the genomic clone λGφ5 (see Example 4). PCRamplification was carried out using the forward primer TCP1.57 pairedindividually with the reverse primers TCP1.46, TCP1.48, TCP1.50,TCP1.52, TCP1.54, TCP1.56, and TCP1.58 (see Table 2). The products fromgenomic DNA of the TCP1.57/TCP1.46, TCP1.48, TCP1.50, TCP1.52, TCP1.54,or TCP1.56 amplifications were approximately 100 basepairs larger thanthe products of the pGRN121 amplifications. The TCP1.57/TCP1.58amplification was the same on either genomic or pGRN121 DNA. Thisindicated the genomic DNA contained an insertion between the sites forTCP1.58 and TCP1.50. The PCR products of TCP1.57/TCP1.50 andTCP1.57/TCP1.52 were sequenced directly, without subcloning, using theprimers TCP1.39, TCP1.57, and TCP1.49.

[0797] As shown below, the 104-base intronic sequence SEQ ID NO:7 isinserted in the hTRT mRNA (shown in bold) at the junction correspondingto bases 274 and 275 of FIG. 16: CCCCCCGCCGCCCCCTCCTTCCGCCAG/GTGGGC (SEQID NO:599) CTCCCCGGGGTCGGCGTCCGGCTGGGGTTGAGGGCGGCCGGGGGGAACCAGCGACATGCGGAGAGCAG CGCAGGCGACTCAGGGCGCTTCCCCCGCAG/GTGTCCTGCCTGAAGGAGCTGGTGGCCCGAGTGCTGC AG

[0798] The “/” indicates the splice junctions; the sequence shows goodmatches to consensus 5′ and 3′ splice site sequences typical for humanintrons.

[0799] This intron contains motifs characteristic of a topoisomerase IIcleavage site and a NFκB binding site (see FIG. 21). These motifs are ofinterest, in part, because expression of topoisomerase II is upregulated in most tumors. It functions to relax DNA by cutting andrewinding the DNA, thus increasing expression of particular genes.Inhibitors of topoisomerase II have been shown to work as anti-tumoragents. In the case of NFκB, this transcription factor may play a rolein regulation of telomerase during terminal differentiation, such as inearly repression of telomerase during development and so is anothertarget for therapeutic intervention to regulate telomerase activity incells.

Example 4 Cloning of Lambda Phage GΦ5 and Characterization of hTRTGenomic Sequences

[0800] A. Lambda GΦ5

[0801] A human genomic DNA library was screened by PCR and hybridizationto identify a genomic clone containing hTRT RNA coding sequences. Thelibrary was a human fibroblast genomic library made using DNA from WI38lung fibroblast cells (Stratagene, Cat # 946204). In this library,partial Sau3AI fragments are ligated into the XhoI site of Lambda FIX711Vector (Stratagene), with an insert size of 9-22 kb.

[0802] The genomic library was divided into pools of 150,000 phage each,and each pool screened by nested PCR (outer primer pair TCP1.52 &TCP1.57; inner pair TCP1.49 & TCP1.50, see Table 1). These primer pairsspan a putative intron (see Example 3, supra) in the genomic DNA of hTRTand ensured the PCR product was derived from a genomic source and notfrom contamination by the hTRT cDNA clone. Positive pools were furthersubdivided until a pool of 2000 phage was obtained. This pool was platedat low density and screened via hybridization with a DNA fragmentencompassing basepairs 1552-2108 of FIG. 16 (restriction sites SphI andEcoRV, respectively).

[0803] Two positive clones were isolated and rescreened via nested PCRas described above; both clones were positive by PCR. One of the clones(λGΦ5) was digested with NotI, revealing an insert size of approximately20 kb. Subsequent mapping (see below) indicated the insert size was 15kb and that phage GΦ5 contains approximately 13 kb of DNA upstream fromthe start site of the cDNA sequence.

[0804] Phage GΦ5 was mapped by restriction enzyme digestion and DNAsequencing. The resulting map is shown in FIG. 7. The phage DNA wasdigested with NcoI and the fragments cloned into pBBS167. The resultingsubclones were screened by PCR to identify those containing sequencecorresponding to the 5′ region of the hTRT cDNA. A subclone (pGRN140)containing a 9 kb NcoI fragment (with hTRT gene sequence and 4-5 kb oflambda vector sequence) was partially sequenced to determine theorientation of the insert. pGRN 140 was digested using SalI to removelambda vector sequences, resulting in pGRN144. pGRN144 was thensequenced. The results of the sequencing are provided in FIG. 21. The 5′end of the hTRT mRNA corresponds to base 2441 of FIG. 21. As indicatedin FIG. 7, two Alu sequence elements are located 1700 base pairsupstream of the hTRT cDNA 5′ end and provide a likely upstream limit tothe promoter region of hTRT. The sequence also reveals an intronpositioned at bases 4173 in FIG. 21, 3′ to the intron described inExample 3, supra.

[0805] B. Additional Genomic Clones

[0806] In addition to the genomic clone described above, two P1bacteriophage clones and one human BAC clone are provided asillustrative embodiments of the invention. P1 inserts are usually 75-100kb, and BAC inserts are usually over 100 Kb.

[0807] The P1 clones (DMPC-HFF#1-477(F6)-GS #15371 andDMPC-HEF#1-1103(H6)-GS #15372) were obtained by PCR screening of a humanP1 library derived from human foreskin fibroblast cells (Shepherd etal., 1994, PNAS USA 91:2629) using primers TCP1.12 and UTR2 whichamplify the 3′ end of hTRT. These clones were both negative (failed toamplify) with primers that amplify the 5′ end of hTRT.

[0808] The human BAC clone (326 E 20) was obtained with a hybridizationscreen of a BAC human genomic library using an 1143 bp Sph1/Xmn1fragment of pGRN121 (FIG. 16; bases 1552-2695) that encompasses the RTmotif region. The clone is believed to include the 5′ end of the gene.The hTRT genomic clones in this example are believed to encompass theentire hTRT gene.

Example 5 Chromosomal Location of hTRT Gene

[0809] The hTRT gene was localized to chromosome 5p by radiation hybridmapping (Boehnke et al., 1991, Am J Hum Genet 49:1174; Walter et al.,1994, Nature Genet 7:22) using the medium resolution Stanford G3 panelof 83 RH clones of the whole human genome (created at the Stanford HumanGenome Center). A human lymphoblastoid cell line (donor; rM) was exposedto 10,000 rad of x-rays and was then fused with nonirradiated hamsterrecipient cells (A3). Eighty-three independent somatic cell hybridclones were isolated, and each represents a fusion event between anirradiated donor cell and a recipient hamster cell. The panel of G3 DNAwas used for ordering markers in the region of interest as well asestablishing the distance between these markers.

[0810] The primers used for the RH mapping were TCP1.12 and UTR2 withamplification conditions of 94° C. 45 sec, 55° C. 45 sec, 72° C. 45 sec,for 45 cycles using Boehringer Mannheim Taq buffer and Perkin-Elmer Taq.The 83 pools were amplified independently and 14 (17%) scored positivefor hTRT (by appearance of a 346 bp band). The amplification resultswere submitted to Stanford RH server, which then provided the maplocation, 5p, and the closest marker, STS D5S678.

[0811] By querying the Genethon genome mapping web site, the maplocation identified a YAC that contains the STS marker D5S678: CEPH YAC780_C_(—)3 Size: 390,660 kb. This YAC also contained chromosome 17markers. This result indicated that the hTRT gene is on chromosome 5,near the telomeric end. There are increased copy numbers of 5p in anumber of tumors. Cri-du-chat syndrome also has been mapped to deletionsin this region.

Example 6 Design and Construction of Vectors for Expression of hTRTProteins and Polynucleotides

[0812] Expression of hTRT in Bacteria

[0813] The following portion of this example details the design ofhTRT-expressing bacterial and eukaryotic cell expression vectors toproduce large quantities of full-length, biologically active hTRT.Generation of biologically active hTRT protein in this manner is usefulfor telomerase reconstitution assays, assaying for telomerase activitymodulators, analysis of the activity of newly isolated species of hTRT,identifying and isolating compounds which specifically associate withhTRT, analysis of the activity of an hTRT variant protein that has beensite-specifically mutated, and as an immunogen, as a few examples.

[0814] pThioHis A/hTRT Bacterial Expression Vector

[0815] To produce large quantities of full-length hTRT, the bacterialexpression vector pThioHis A (Invitrogen, San Diego, Calif.) wasselected as an expression system. The hTRT-coding insert includesnucleotides 707 to 4776 of the hTRT insert in the plasmid pGRN121. Thisnucleotide sequence includes the complete coding sequence for the hTRTprotein.

[0816] This expression vector of the invention is designed for inducibleexpression in bacteria. The vector can be induced to express, in E.coli, high levels of a fusion protein composed of a cleavable, HIStagged thioredoxin moiety and the full length hTRT protein. The use ofthe expression system was in substantial accordance with themanufacturer's instructions. The amino acid sequence of the fusionprotein encoded by the resulting vector of the invention is shown below;(-*-) denotes an enterokinase cleavage site:MSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCG (SEQ ID NO:600)PCKMIAPILDEIADEYQGKLTVAKLRIDHNPGTA PKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGDDDDK-*-VPMHELEIFEFAAA STQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFR ALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFT TSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQA RPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVG QGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLRPLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPW DTPCPPVYAETKHFLYSSGDKEQSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQ MRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHS SPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSP GVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQH LKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKA LFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEV IASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSS SLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLL RLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCG LLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTN IYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSE AVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

[0817] pGEX-2TK with hTRT Nucleotides 3272 to 4177 of pGRN121

[0818] This construct of the invention is used to produce fusion proteinfor, e.g., the purpose of raising polyclonal and monoclonal antibodiesto hTRT protein. Fragments of hTRT can also be used for other purposes,such as to modulate telomerase activity, for example, as adominant-negative mutant or to prevent the association of a telomerasecomponent with other proteins or nucleic acids.

[0819] To produce large quantities of an hTRT protein fragment, the E.coli expression vector pGEX-2TK (Pharmacia Biotech, Piscataway N.J.) wasselected, and used essentially according to manufacturer's instructionsto make an expression vector of the invention. The resulting constructcontains an insert derived from nucleotides 3272 to 4177 of the hTRTinsert in the plasmid pGRN121. The vector directs expression in E. coliof high levels of a fusion protein composed of glutathione-S-transferasesequence (underlined below), thrombin cleavage sequence (doubleunderlined), recognition sequence for heart muscle protein kinase(italicized), residues introduced by cloning in brackets ([GSVTK]; SEQID NO:601) and hTRT protein fragment (in bold) as shown below:MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYE (SEQ ID NO:602)RDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDR LCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQ GWQATFGGGDHPPKSD LVPRGS RRASV[GSVTK]IPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLL RLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCG LLLDTRTLEVQSDYSSYARTSIRASVTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTN IYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSE AVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

[0820] When this fusion protein was expressed, it formed insolubleaggregates. It was treated generally as described above, in the sectionentitled purification of proteins from inclusion bodies. Specifically,induced cells were suspended in PBS (20 mM sodium phosphate, pH 7.4, 150mM NaCl) and disrupted by sonication. NP-40 was added to 0.1%, and themixture was incubated for 30 minutes at 4° C. with gentle mixing. Theinsoluble material was collected by centrifugation at 25,000 g for 30minutes at 4° C. The insoluble material was washed once in 4M urea inPBS, collected by centrifugation, then washed again in PBS. Thecollected pellet was estimated to contain greater than 75% fusionprotein. This material was dried in a speed vacuum, then suspended inadjuvant for injection into mice and rabbits for the generation ofantibodies. Separation of the recombinant protein from the glutathioneS-transferase moiety is accomplished by site-specific proteolysis usingthrombin according to manufacturer's instructions.

[0821] pGEX-2TK with hTRT Nucleotides 2426 to 3274 of pGRN121 with HIS-8Tag

[0822] To produce large quantities of a fragment of hTRT, another E.coli expression vector pGEX-2TK construct was prepared. This constructcontains an insert derived from nucleotides 2426 to 3274 of the hTRTinsert in the plasmid pGRN121 and a sequence encoding eight consecutivehistidine residues (HIS-8 Tag). To insert the HIS-8 TAG, the pGEX-2TKvector with hTRT nucleotides 2426 to 3274 of pGRN121 was linearized withBamH1. This opened the plasmid at the junction between theGST-thrombin-heart muscle protein kinase and the hTRT coding sequence. Adouble stranded oligonucleotide with BamH1 compatible ends was ligatedto the linearized plasmid resulting in the in-frame introduction ofeight histidine residues upstream of the hTRT sequence.

[0823] The vector directs expression in E coli of high levels of afusion protein composed of glutathione-S-transferase sequence(underlined); thrombin cleavage sequence (double underlined);recognition sequence for heart muscle protein kinase (italicized); a setof three and a set of five residues introduced by cloning are inbrackets ([GSV] and [GSVTK] SEQ ID NO:601); eight consecutive histidines(also double underlined); and hTRT protein fragment (in bold):MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYE (SEQ ID NO:603)RDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDR LCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQ GWQATFGGGDHPPKSD LVPRGS RRASV[GSV]HHHHHHHH[GSVTK]MSVYVVELLRSFFYVTETTFQ KNRLFFYRPSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMD YVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPE LYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYM RQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGI

[0824] Each of the pGEX-2TK vectors of the invention can be used toproduce fusion protein for the purpose of raising polyclonal andmonoclonal antibodies to hTRT protein. Additionally, this fusion proteincan be used to affinity purify antibodies raised to hTRT peptides thatare encompassed within the fusion protein. Separation of the recombinantprotein from the glutathione S-transferase moiety can be accomplished bysite-specific proteolysis using thrombin according to manufacturer'sinstructions.

[0825] pGEX-2TK with hTRT Nucleotides 2426 to 3274 of pGRN121, no HIS-8Tag

[0826] To produce large quantities of a fragment of hTRT, another E.coli expression vector pGEX-2TK construct was prepared.

[0827] This construct contains an insert derived from nucleotides 2426to 3274 of the hTRT insert in the plasmid pGRN121, but without the HIS-8tag of the construct described above. The vector directs expression in Ecoli of high levels of a fusion protein composed ofglutathione-S-transferase (underlined), thrombin cleavage sequence(double underlined), recognition sequence for heart muscle proteinkinase (italicized), residues introduced by cloning in brackets([GSVTK]; SEQ ID NO:601) and hTRT protein fragment (in bold):MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYE (SEQ ID NO:604)RDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDR TCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQ GWQATFGGGDHPPKSD LVPRGS RRASV[GSVTK]MSVYVVELLRSFFYVTETTFQKNRLFFYRPSVWS KLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKR AERLTSRKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPEYFVKVDVTGAYDTI PQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDA VVIEQSSSLNEASGLFDVFLRFMCHHAVRIRGKSYVQCQGI

[0828] pGEX-2TK with hTRT Nucleotides 1625 to 2458 of pGRN121

[0829] To produce large quantities of a fragment of hTRT protein,another E. coli expression vector pGEX-2TK construct was prepared.

[0830] This construct contains an insert derived from nucleotides 1625to 2458 of the hTRT insert in the plasmid pGRN121. The vector directsexpression in E coli of high levels of a fusion protein composed ofglutathione-S-transferase, (underlined), thrombin cleavage sequence(double underlined), recognition sequence for heart muscle proteinkinase (italicized) residues introduced by cloning in brackets ([GSVTK];SEQ ID NO:601) and hTRT protein fragment (in bold):MSPILGYWKIKGLVOPTRLLLEYLEEKYEEHLYE (SEQ ID NO:605)RDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDR LCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQ

ATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPR PWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLP QRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQL LRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAW LRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRS

[0831] pGEX-2TK with hTRT Nucleotides 782 to 1636 of pGRN121

[0832] To produce large quantities of a fragment of hTRT protein,another E. coli expression vector pGEX-2TK construct was prepared.

[0833] This construct contains an insert derived from nucleotides 782 to1636 of the hTRT insert in the plasmid pGRN121. The vector directsexpression in E coli of high levels of a fusion protein composed ofglutathione-S-transferase, (underlined), thrombin cleavage sequence(double underlined), recognition sequence for heart muscle proteinkinase (italicized) residues introduced by cloning in brackets ([GSVTK];SEQ ID NO:601) and hTRT protein fragment (in bold):MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYE (SEQ ID NO:606)RDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQS MAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDR LCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQ GWQATFGGGDHPPKSD LVPRGS RRASV[GSVTK]MPRAPRCRAVRSLLSHYREVLPLATFVRRLGPQG WRLVQRGDPAAFRALVAQCLVCVPWDARPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALL DGARGGPPEATTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPCAYQVCGPP LYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGA APEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSL

[0834] pT7FLhTRT with hTRT cDNA Lacking 5′-Non-Coding Sequence

[0835] As described above, in one embodiment, the invention provides foran hTRT that is modified in a site-specific manner to facilitate cloninginto bacterial, mammalian, yeast and insect expression vectors withoutany 5′ untranslated hTRT sequence. In some circumstances, minimizing theamount of non-protein encoding sequence allows for improved proteinproduction (yield) and increased mRNA stability. In this embodiment ofthe invention, the hTRT gene's 5′ non-coding region was removed beforecloning into a bacterial expression vector.

[0836] This was effected by engineering an additional restrictionendonuclease site just upstream (5′) to the start (ATG) codon of thehTRT coding sequence (FIG. 16). The creation of a restriction site just5′ to the coding region of the protein allows for efficient productionof a wide variety of vectors that encode fusion proteins, such as fusionproteins comprising labels and peptide TAGs, for immunodetection andpurification.

[0837] Specifically, the oligonucleotide5′-CCGGCCACCCCCCATATGCCGCGCGCTCCC-3′ (SEQ ID NO:607) was used asdescribed above to modify hTRT cDNA nucleotides 779 to 781 of the hTRTcDNA (FIG. 16) from GCG to CAT. These 3 nucleotides are the lastnucleotides before the ATG start codon so they do not modify the proteinsequence. The change in sequence results in the creation of a uniqueNdeI restriction site in the hTRT cDNA. Single-stranded hTRT DNA wasused as a DNA source for the site directed mutagenesis. The resultingplasmid was sequenced to confirm the success of the mutagenesis.

[0838] This modification allowed the construction of the followingplasmid of the invention, designated pT7FLhTRT. The site-specificallymodified hTRT sequence (addition of the NdeI restriction site) wasdigested with NdeI and NotI (and filled in with Klenow enzyme togenerate blunt ended DNA) to generate an hTRT encoding nucleic acidfragment. The fragment was then cloned into a pSL3418 plasmid previouslyrestriction digested with NdeI and SmaI (also a blunt ended cutter).pSL3418 is a modified pAED4 plasmid into which a FLAG sequence (ImmunexCorp, Seattle Wash.) and an enterokinase sequence are inserted justupstream from the above-referenced NdeI site. This plasmid, designatedpT7FLhTR, allows the expression of full length hTRT (with a Flag-Tag atits 5′ end) in an E. coli strain expressing the T7 RNA polymerase.

[0839] Plasmids with hTRT cDNA Lacking 3′-Non-Coding Sequence

[0840] As discussed above, the invention provides for expression vectorscontaining TRT-encoding nucleic acids in which some or all non-codingsequences have been deleted. In some circumstances, minimizing theamount of non-protein encoding sequence allows for improved proteinproduction (yield) and increases mRNA stability. In this embodiment ofthe invention, the 3′ untranslated region of hTRT is deleted beforecloning into a bacterial expression plasmid.

[0841] The plasmid pGRN121, containing the full length hTRT cDNA, asdiscussed above, was first deleted of all Apa1 sites. This was followedby deletion of the MscI-HincII hTRT restriction digest enzyme fragmentcontaining the 3′UTR. The NcoI-XbaI restriction digest fragmentcontaining the stop codon of hTRT was then inserted into the NcoI-XbaIsite of pGRN121 to make a plasmid equivalent to pGRN121, designatedpGRN124, except lacking the 3′UTR.

[0842] Bacterial Expression Vectors Using Antibiotic Selection Markers

[0843] The invention also provides for bacterial expression vectors thatcan contain selection markers to confer a selectable phenotype ontransformed cells and sequences coding for episomal maintenance andreplication such that integration into the host genome is not required.For example, the marker may encode antibiotic resistance, particularlyresistance to chloramphenicol (see Harrod (1997) Nucleic Acids Res. 25:1720-1726), kanamycin, G418, bleomycin and hygromycin, to permitselection of those cells transformed with the desired DNA sequences, seefor example, Blondelet-Rouault (1997) Gene 190:315-317; and Mahan (1995)Proc Natl Acad Sci USA 92:669-673.

[0844] In one embodiment of the invention, the full length hTRT wascloned into a modified BlueScript plasmid vector (Stratagene, San Diego,Calif.), designated pBBS235, into which a chloramphenicol antibioticresistence gene had been inserted. The NotI fragment from pGRN124(discussed above) containing the hTRT ORF into the NotI site of pBBS235so that the TRT ORF is in the opposite orientation of the vector's Lacpromoter. This makes a plasmid that is suitable for mutageneis ofplasmid inserts, such as TRT nucleic acids of the invention. Thisplasmid construct, designated pGRN125, can be used in the methods of theinvention involving mutagenesis of telomerase enzyme and TRT proteincoding sequences and for in vitro transcription of hTRT using the T7promoter (and in vitro transcription of antisense hTRT using the T3promoter).

[0845] In another embodiment of the invention, NotI restriction digestfragments from pGRN124 containing the hTRT ORF were subcloned into theNotI site of pBBS235 (described above) so the TRT ORF is in the sameorientation as the vector's Lac promoter. This makes a plasmid,designated pGRN126, that can be used for expression of full length hTRTin E. coli. The expressed product will contain 29 amino acids encoded bythe vector pBBS235, followed by 18 amino acids encoded by the 5′UTR ofhTRT, followed by the full length hTRT protein.

[0846] In a further embodiment of the invention, in vitro mutagenesis ofpGRN125 was done to convert the hTRT initiating ATG codon into a Kozakconsensus and create EcoRI and BglII restriction digest sites tofacilitate cloning into expression vectors. The oligonucleotide5′-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3′ (SEQ IDNO:608) (altered nucleotides in lower case) was used in the mutagenesisprocedure. The resulting expression vector was designated pGRN127.

[0847] In another embodiment of the invention, the second Asp of the TRT“DD motif” was converted to an alanine to create a non-functionaltelomerse enzyme, thus creating a mutant TRT protein for use as adominant/negative mutant. The hTRT coding sequence was mutagenized invitro using the oligonucleotide5′-CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCT CACC-3′ (SEQID NO:609) to convert the asparagine codon for residue 869 (Asp869) toan alanine (Ala) codon. This also created an MluI restriction enzymesite. The resulting expression plasmid was designated pGRN130, whichalso contains the Kozak consensus sequence as described for pGRN127.

[0848] The invention also provides a vector designed to express anantisense sequence fragment of hTRT. The pGRN126 plasmid was cut tocompletion with MscI and SmaI restriction enzymes and religated todelete over 95% of the hTRT ORF. One SmaI-MscI fragment was re-insertedduring the process to recreate CAT activity. This unpurified plasmid wasthen redigested with SalI and EcoRI and the fragment containing theinitiating codon of the hTRT ORF was inserted into the SalI-EcoRI sitesof pBBS212 to make an antisense expression plasmid expressing theantisense sequence spanning the 5′UTR and 73 bases pair residues of thehTRT ORF (in mammalian cells). This plasmid was designated pGRN135.

[0849] Expression of hTRT Telomerase in Yeast

[0850] The present invention also provides hTRT-expressing yeastexpression vectors to produce large quantities of full-length,biologically active hTRT.

[0851]Pichia pastoris Expression Vector pPICZ B and Full Length hTRT

[0852] To produce large quantities of full-length, biologically activehTRT, the Picha pastoris expression vector pPICZ B (Invitrogen, SanDiego, Calif.) was selected. The hTRT-coding sequence insert was derivedfrom nucleotides 659 to 4801 of the hTRT insert in plasmid pGRN121. Thisnucleotide sequence includes the full-length sequence encoding hTRT.This expression vector is designed for inducible expression in P.pastoris of high levels of full-length, unmodified hTRT protein.Expression is driven by a yeast promoter, but the expressed sequenceutilizes the hTRT initiation and termination codons. No exogenous codonswere introduced by the cloning. The resulting pPICZ B/hTRT vector wasused to transform the yeast.

[0853]Pichia pastoris Expression Vector hTRT-His6/pPICZ B

[0854] A second Picha pastoris expression vector of the inventionderived from pPICZ B, also contains the full-length sequence encodinghTRT derived from nucleotides 659 to 4801 of the hTRT insert in theplasmid pGRN121. This hTRT-His6/pPICZ B expression vector encodes fulllength hTRT protein fused at its C-terminus to the Myc epitope and His6reporter tag sequences. The hTRT stop codon has been removed andreplaced by vector sequences encoding the Myc epitope and the His6reporter tag as well as a stop codon. This vector is designed to directhigh-level inducible expression in yeast of the following fusionprotein, which consists of hTRT sequence (underlined), vector sequencesin brackets ([L] and [NSAVD]; SEQ ID NO:610) the Myc epitope (doubleunderlined), and the His6 tag (italicized):MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQ (SEQ ID NO:611)GWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAA PSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGA WGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWN HSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCV VSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQ LRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVL LKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPG LWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKF LHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREA RPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASV LGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQ KAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHH AVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDLLLRLVDDFLLVTPHLTHAKTFLR TLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTS IRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPF HQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTY VPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD[L]EQKLISEEDL[NSAVD]HHHHH H

[0855] Expression of hTRT in Insect Cells

[0856] The present invention also provides hTRT telomerase-expressinginsect cell expression vectors that produce large quantities offull-length, biologically active hTRT.

[0857] Baculovirus Expression Vector pVL1393 and Full Length hTRT

[0858] The telomerase coding sequence of interest was cloned into thebaculovirus expression vector pVL1393 (Invitrogen, San Diego, Calif.).This construct was subsequently cotransfected into Spodoptera fungupeida(sf-9) cells with linearized DNA from Autograph california nuclearpolyhedrosis virus (Baculogold-AcMNPV). The recombinant baculovirusesobtained were subsequently plaque purified and expanded followingstandard protocols.

[0859] This expression vector provides for expression in insect cells ofhigh levels of full-length hTRT protein. Expression is driven by abaculoviral polyhedrin gene promoter. No exogenous codons wereintroduced by the cloning.

[0860] Baculovirus Expression Vector pBlueBacHis2 B and Full Length hTRT

[0861] To produce large quantities of full-length, biologically activehTRT, the baculovirus expression vector pBlueBacHis2 B (Invitrogen, SanDiego, Calif.) was selected as a source of control elements. ThehTRT-coding insert consisted of nucleotides 707 to 4776 of the hTRTinsert in plasmid pGRN121.

[0862] A full length hTRT with a His6 and Anti-Xpress tags (Invitrogen)was also constructed. This vector also contains an insert consisting ofnucleotides 707 to 4776 of the hTRT insert from the plasmid pGRN121. Thevector directs expression in insect cells of high levels of full lengthhTRT protein fused to a cleavable 6-histidine and Anti-Xpress tags, andthe amino acid sequence of the fusion protein is shown below; (-*-)denotes enterokinase cleavage site:      MPRGSHHHHHHGMASMTGGQQMGRDLYDD(SEQ ID NO:612) DDL-*-DPSSRSAAGTMEFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLAT FVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAK NVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLV APSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASR SLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPS VGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFL GSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGS VAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAK LSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQK NRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDY VVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPEL YFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMR QFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLC SLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVE DEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLR RLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSI LKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTA LEAAANPALPSDFKTILD

[0863] Baculovirus Expression Vector pBlueBac4.5 and Full Length h TRTProtein

[0864] To produce large quantities of full-length, biologically activehTRT, a second baculovirus expression vector, pBlueBac4.5 (Invitrogen,San Diego, Calif.) was constructed. The hTRT-coding insert alsoconsisted of nucleotides 707 to 4776 of the hTRT from the plasmidpGRN121.

[0865] Baculovirus Expression Vector pMelBacB and Full Length hTRTProtein

[0866] To produce large quantities of full-length, biologically activehTRT, a third baculovirus expression vector, pMelBacB (Invitrogen, SanDiego, Calif.) was constructed. The hTRT-coding insert also consists ofnucleotides 707 to 4776 of the hTRT insert from the plasmid pGRN121.

[0867] pMelBacB directs expression of full length hTRT in insect cellsto the extracellular medium through the secretory pathway using themelittin signal sequence. High levels of full length hTRT are thussecreted. The melittin signal sequence is cleaved upon excretion, but ispart of the protein pool that remains intracellularly. For that reason,it is indicated in parentheses in the following sequence. The sequenceof the fusion protein encoded by the vector is shown below:    (MKFLVNVALVFMVVYISYIYA)-*-DPSS (SEQ ID NO:613)RSAAGTMEFAAASTQRCVLLRTWEALAPATPAMP RAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPS FRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWG LLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHS VREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVS PARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLR PSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLK THCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLW GSRHNERRFLRNTKKFISLGHKAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLH WLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARP ALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLG LDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKA AHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAV RIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRT LVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSI RASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFH QQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYV PLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

[0868] Expression of hTRT in Mammalian Cells

[0869] The present invention also provides vectors to produce hTRT inlarge quantities as full-length, biologically active protein in avariety of mammalian cell lines, which is useful in many embodiments ofthe invention, as discussed above.

[0870] MPSV-hTRT Expression Plasmids

[0871] The invention also provides for an expression system for use inmammalian cells that gives the highest possible expression ofrecombinant protein, such as telomerase, without actually modifying thecoding sequence (e.g. optimizing codon usage). In one embodiment, theinvention provides MPSV mammalian expression plasmids (from plasmidpBBS212, described as pMPSV-TM in Lin J-H (1994) Gene 47:287-292)capable of expressing the TRTs of the invention. The MPSV plasmids canbe expressed either as stable or transient clones.

[0872] In this expression system, while the hTRT coding sequence itselfis unchanged, exogenous transcriptional control elements areincorporated into the vector. The myeloproliferative sarcoma virus(MPSV) LTR (MPSV-LTR) promoter, enhanced by the cytomegalovirus (CMV)enhancer, is incorporated for transcriptional initiation. This promoterconsistently shows higher expression levels in cell lines (see Lin J-H(1994) supra). A Kozak consensus sequence can be incorporated fortranslation initiation (see Kozak (1996) Mamm. Genome 7:563-574). Allextraneous 5′ and 3′ untranslated hTRT sequences can be removed toinsure that these sequences do not interfere with expression, asdiscussed above. The MPSV plasmid containing the complete hTRT codingsequence, with all extraneous sequences included, is designated pGRN133.A control, hTRT “antisense” plasmid was also constructed. This vector isidentical to pGRN133 except that the TRT insert is the antisensesequence of hTRT (the antisense, which control can be used as a vectoris designated pGRN134). The MPSV plasmid containing the complete hTRTcoding sequence with all other extraneous sequences removed andcontaining the Kozak consensus sequence is designated pGRN145.

[0873] Two selection markers, PAC(Puromycin-N-acetyl-transferase=Puromycin resistance) and HygB(Hygromycin B=Hygromycin resistance) are present for selection of theplasmids after transfection (see discussion referring to selectablemarkers, above). Double selection using markers on both sides of thevector polylinker should increase the stability of the hTRT codingsequence. A DHFR (dihydrofolate reductase) encoding sequence is includedto allow amplification of the expression cassette after stable clonesare made. Other means of gene amplification can also be used to increaserecombinant protein yields.

[0874] The invention also provides for MPSV mammalian expressionplasmids containing hTRT fusion proteins. In one embodiment, the hTRTsequence, while retaining its 5′ untranslated region, is linked to anepitope flag, such as the IBI FLAG (International Biotechnologies Inc.(IBI), Kodak, New Haven, Conn.) and inserted into the MPSV expressionplasmid (designated pGRN147). This particular constuct contains a Kozaktranslation initiation site. The expressed fusion protein can bepurified using the M-1 anti-FLAG octapeptide monoclonal antibody (IBI,Kodak, supra).

[0875] In another embodiment, hTRT is site-specifically altered. Oneamino acid residue codon is mutagenized, changing the aspartic acid atposition 869 to an alanine. This Asp869->Ala hTRT mutant, retaining its5′ untranslated region and incorporating a Kozak sequence, was insertedinto an MPSV expression plasmid, and designated pGRN146. The Asp869->AlahTRT mutant was further engineered to contain the FLAG sequence, asdescribed above, and the insert cloned into an MPSV expression plasmid.One such expression plasmid is designated pGRN154-I. Specifically, forpGRN154-I, an Eam1105I restriction digest fragment from pGRN146containing the Kozak sequence-containing “front end” (5′ segment) ofhTRT is cloned into the Eam1105I sites of pGRN147 (see above) to make anMPSV expression plasmid capable of expressing hTRT with a Kozaksequence, the above-described D869->A mutation, and the IBI flag.

[0876] Another embodiment of the invention is an expression plasmidderived from pGRN146. The mammalian expression plasmid, designatedpGRN152, was generated by excising the EcoRI fragment from plasmidpGRN146 (containing the hTRT ORF) and cloned into the EcoRI site ofpBBS212 to remove the 5′UTR of hTRT. The hTRT is oriented so that itsexpression is controlled by the MPSV promoter. This makes a mammalianexpression plasmid that expresses hTRT with a Kozak consensus sequenceand the D869->A mutation, and uses the MPSV promoter.

[0877] The invention provides for a mammalian expression vector in whichhTRT is oriented so that the hTRT coding sequence is driven by the MPSVpromoter. For example, an EcoR1 restriction digest fragment from pGRN137containing the hTRT open reading frame (ORF) was cloned into the EcoR1site of pBBS212 (see below), thus removing the 5′ untranslated region(5′-UTR) of hTRT. pGRN137 was constructed by excising a SalI-Sse8387Ifragment from pGRN130, described below, containing the Kozak mutation ofhTRT into the Sal 1-SSE 83871 sites of pGRN136, making a mammalianexpression plasmid expressing hTRT containing a Kozak consensus sequenceoff the MPSV promoter. Plasmid pGRN136 was constructed by excising aHindIII SalI fragment from pGRN126 containing the hTRT ORF and cloningit into the HindIII SalI sites of plasmid, pBBS242, making a mammalianexpression plasmid expressing hTRT off the MPSV promoter). This makes amammalian expression plasmid, designated pGRN145, that expresses hTRTwith a Kozak consensus sequence using the MPSV promoter. See also thepGRN152 MPSV promoter-driven mammalian expression vector describedbelow.

[0878] hTRT Expressed in 293 Cells using Episomal Vector pEBVHis

[0879] An episomal vector, pEBVHis (Invitrogen, San Diego, Calif.) wasengineered to express an hTRT fusion protein comprising hTRT fused to anN-terminal extension epitope tag, the Xpress epitope (Invitrogen, SanDiego, Calif.) (designated pGRN122). The NotI hTRT fragment from pGRN121containing the hTRT ORF was cloned into the NotI site of pEBVHisA sothat the hTRT ORF is in the same orientation as the vector's RousSarcoma Virus (RSV) promoter. In this orientation the His6 flag wasrelatively closer to the N-terminus of hTRT.

[0880] A vector was also constructed containing as an insert theantisense sequence of hTRT and the epitope tag (the plasmid designatedpGRN123, which can be used as a control). The vector was transfectedinto 293 cells and translated hTRT identified and isolated using anantibody specific for the Xpress epitope. pEBVHis is a hygromycinresistant EBV episomal vector that expresses the protein of interestfused to a N-terminal peptide. Cells carrying the vector are selectedand expanded, then nuclear and cytoplasmic extracts prepared. These andcontrol extracts are immunoprecipitated with anti-Xpress antibody, andthe immunoprecipitated beads are tested for telomerase activity byconventional assay.

[0881] Expression of Recombinant hTRT in Mortal, Normal Diploid HumanCells

[0882] In one embodiment of the invention, recombinant hTRT andnecessary telomerase enzyme complex components can be expressed innormal, diploid mortal cells to increase their proliferative capacity orto immortalize them, or to facilitate immortalizing them. This allowsone to obtain diploid immortal cells with an otherwise normal phenotypeand karotype. As discussed above, this use of telomerase has enormouscommercial utility.

[0883] Sense hTRT (FIG. 16) and antisense hTRT were cloned into a CMVvector. These vectors were purified and transiently transfected into twonormal, mortal, diploid human cell clones. The human clones were youngpassage diploid human BJ and IMR90 cell strains.

[0884] Analysis of telomerase activity using a TRAP assay utilizing theTRAPeze™ Kit (Oncor, Inc., Gaithersburg, Md.) showed that transfectionof sense hTRT—but not antisense hTRT—generated telomerase activity inboth the BJ and IMR90 cell strains.

[0885] Expression of Recombinant hTRT in Immoralized IMR90 Human Cells

[0886] Using the same hTRT sense construct cloned into CMV vectors usedin the above described diploid human BJ and IMR90 cell strains studies,immortalized SW13 ALT pathway cell line (an IMR90 cell immortalized withSV40 antigen) was transiently transfected. A TRAP assay (TRAPeze, Oncor,Inc, Gaithersburg, Md.) demonstrated that telomerase activity wasgenerated in the sense construct transfected cells.

[0887] Vectors for Regulated Expression of hTRT in Mammalian Cells:Inducible and Repressible Expression of hTRT

[0888] The invention provides vectors that can be manipulated to induceor repress the expression of the TRTs of the invention, such as hTRT.For example, the hTRT coding sequence can be cloned into theEcdysone-Inducible Expression System from Invitrogen (San Diego, Calif.)and the Tet-On and Tet-off tetracycline regulated systems from ClontechLaboratories, Inc. (Palo Alto, Calif.). Such inducible expressionsystems are provided for use in the methods of the invention where it isimportant to control the level or rate of transcription of transfectedTRT. For example, the invention provides for cell lines immortalizedthrough the expression of hTRT; such cells can be rendered “mortal” byinhibition of hTRT expression by the vector through transcriptionalcontrols, such as those provided by the Tet-Off system. The inventionalso provides for methods of expressing TRT only transiently to avoidthe constitutive expression of hTRT, which may lead to unwanted“immortalization” of the transfected cells, as discussed above.

[0889] The Ecdysone-Inducible Mammalian Expression System is designed toallow regulated expression of the gene of interest in mammalian cells.The system is distinguished by its tightly regulated mechanism thatallows almost no detectable basal expression and greater than 200-foldinducibility in mammalian cells. The expression system is based on theheterodimeric ecdysone receptor of Drosophila. The Ecdysone-InducibleExpression System uses a steroid hormone ecdysone analog, muristerone A,to activate expression of hTRT via a heterodimeric nuclear receptor.Expression levels have been reported to exceed 200-fold over basallevels with no effect on mammalian cell physiology “Ecdysone-InducibleGene Expression in Mammalian Cells and Transgenic Mice” (1996) Proc.Natl. Acad. Sci. USA 93, 3346-3351). Once the receptor binds ecdysone ormuristerone, an analog of ecdysone, the receptor activates anecdysone-responsive promoter to give controlled expression of the geneof interest. In the Ecdysone-Inducible Mammalian Expression System, bothmonomers of the heterodimeric receptor are constitutively expressed fromthe same vector, pVgRXR. The ecdysone-responsive promoter, whichultimately drives expression of the gene of interest, is located on asecond vector, pIND, which drives the transcription of the gene ofinterest.

[0890] The hTRT coding sequence is cloned in the pIND vector (ClontechLaboratories, Inc, Palo Alto, Calif.), which contains 5 modifiedecdysone response elements (E/GREs) upstream of a minimal heat shockpromoter and the multiple cloning site. The construct is thentransfected in cell lines which have been pre-engineered to stablyexpress the ecdysone receptor. After transfection, cells are treatedwith muristerone A to induce intracellular expression from pIND.

[0891] The Tet-on and Tet-off expression systems (Clontech, Palo Alto,Calif.) give access to the regulated, high-level gene expression systemsdescribed by Gossen (1992) “Tight control of gene expression inmammalian cells by tetracycline responsive promoters” Proc. Natl. Acad.Sci. USA 89:5547-5551, for the Tet-Off transcription repression system;and Gossen (1995) “Transcriptional activation by tetracycline inmammalian cells” Science 268:1766-1769, for the Tet-On inducibletranscriptional system. In “Tet-Off” transformed cell lines, geneexpression is turned on when tetracycline (Tc) or doxycycline (“Dox;” aTc derivative) is removed from the culture medium. In contrast,expression is turned on in Tet-On cell lines by the addition of Tc orDox to the medium. Both systems permit expression of cloned genes to beregulated closely in response to varying concentrations of Tc or Dox.

[0892] This system uses the “pTRE” as a response plasmid that can beused to express a gene of interest. Plasmid pTRE contains a multiplecloning site (MCS) immediately downstream of the Tet-responsive PhCMV*-1promoter. Genes or cDNAs of interest inserted into one of the sites inthe MCS will be responsive to the tTA and rtTA regulatory proteins inthe Tet-Off and Tet-On systems, respectively. PhCMV*-1 contains theeTet-responsive element (TRE), which consists of seven copies of the42-bp tet operator sequence (tetO). The TRE element is just upstream ofthe minimal CMV promoter (PminCMV), which lacks the enhancer that ispart of the complete CMV promoter in the pTet plasmids. Consequently,PhCMV*-1 is silent in the absence of binding of regulatory proteins tothe tetO sequences. The cloned insert must have an initiation codon. Insome cases, addition of a Kozak consensus ribosome binding site mayimprove expression levels; however, many cDNAs have been efficientlyexpressed in Tet systems without the addition of a Kozak sequence.pTRE-Gene X plasmids are cotransfected with pTK-Hyg to permit selectionof stable transfectants.

[0893] Setting up a Tet-Off or Tet-On expression system generallyrequires two consecutive stable transfections to create a“double-stable” cell line that contains integrated copies of genesencoding the appropriate regulatory protein and TRT under the control ofa TRE. In the first transfection, the appropriate regulatory protein isintroduced into the cell line of choice by transfection of a “regulatorplasmid” such as pTet-Off or pTet-On vector, which expresses theappropriate regulatory proteins. The hTRT cloned in the pTRE “responseplasmid” is then introduced in the second transfection to create thedouble-stable Tet-Off or Tet-On cell line. Both systems give very tighton/off control of gene expression, regulated dose-dependent induction,and high absolute levels of gene expression.

[0894] Expression Recombinant hTRT with DHFR and Adenovirus Sequences

[0895] The pGRN155 plasmid construct was designed for transientexpression of hTRT cDNA in mammalian cells. A Kozak consensus isinserted at the 5′ end of the hTRT sequence. The hTRT insert contains no3′ or 5′ UTR. The hTRT cDNA is inserted into the EcoRI site of p91023(B)(Wong (1985) Science 228:810-815). The hTRT insert is in the sameorientation as the DHFR ORF.

[0896] Plasmid pGRN155 contains the SV40 origin and enhancer justupstream of an adenovirus promoter, a tetracycline resistance gene, anE. coli origin and an adenovirus VAI and VAII gene region. Thisexpression cassette contains, in the following order: the adenovirusmajor late promoter; the adenovirus tripartite leader; a hybrid intronconsisting of a 5′ splice site from the first exon of the tripartiteleader and a 3′ splice site from the mouse immunoglobulin gene; the hTRTcDNA; the mouse DHFR coding sequence; and, the SV40 polyadenylationsignal.

[0897] The adenovirus tripartite leader and the VA RNAs have beenreported to increase the efficiency with which polycistronic mRNAs aretranslated. DHFR sequences have been reported to enhance the stabilityof hybrid mRNA. DHFR sequences also can provide a marker for selectionand amplification of vector sequences. See Logan (1984) Proc. Natl.Acad. Sci. USA 81:3655); Kaufman (1985) Proc. Natl. Acad. Sci. USA 82:689; and Kaufman (1988) Focus (Life Technologies, Inc.), Vol.10, no. 3).This makes the expression vector particularly useful for transientexpression.

[0898] Other expression plamids of the invention are described forillustrative purposes.

[0899] pGRN121

[0900] The EcoRI fragment from lambda clone 25-1.1.6 containing theentire cDNA encoding hTRT protein was inserted into the EcoRI site ofpBluescriptIISK+ such that the 5′ end of the cDNA is near the T7promoter in the vector. The selectable marker that is used with thisvector is ampicillin.

[0901] pGRN122

[0902] The NotI fragment from pGRN121 containing the hTRT ORF wasinserted into the NotI site of pEBVHisA so that the coding sequence isoperably linked to the RSV promoter. This plasmid expresses a fusionprotein composed of a His6 flag fused to the N-terminal of the hTRTprotein. The selectable marker that is used with this vector isampicillin or hygromycin.

[0903] pGRN123

[0904] The NotI fragment from pGRN121 containing the hTRT ORF wasinserted into the NotI site of pEBVHisA so that the coding sequence isin the opposite orientation as the RSV promoter, thus expressingantisense hTRT.

[0905] pGRN124

[0906] Plasmid pGRN121 was deleted of all ApaI sites followed bydeletion of the MscI-HincII fragment containing the 3′UTR. The Nco-XbaIfragment containing the stop codon of the hTRT coding sequence was theninserted into the Nco-XbaI sites of pGRN121 to make a plasmid equivalentto pGRN121 except lacking the 3′UTR, which may be preferred forincreased expression levels in some cells.

[0907] pGRN125

[0908] The NotI fragment from pGRN124 containing the hTRT codingsequence was inserted into the NotI site of pBBS235 so that the openreading frame is in the opposite orientation of the Lac promoter. Theselectable marker that is used with this vector is chloramphenicol.

[0909] pGRN126

[0910] The NotI fragment from pGRN124 containing the hTRT codingsequence was inserted into the NotI site of pBBS235 so that the hTRTcoding sequence inserted is in the same orientation as the Lac promoter.

[0911] pGRN127

[0912] The oligonucleotide 5′-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3′ (SEQ ID NO:608) was used in in vitro mutagenesis ofpGRN125 to convert the initiating ATG codon of the hTRT coding sequenceinto a Kozak consensus sequence and create EcoRI and BglII sites forcloning. Also, oligonucleotide COD2866 was used to convert AmpS to AmpR(ampicillin resistant) and oligonucleotide COD1941 was used to convertCatR (chloramphenicol resistant) to CatS (chloramphenicol sensitive).

[0913] pGRN128

[0914] The oligonucleotide 5′-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3′ (SEQ ID NO:608) is used in in vitro mutagenesis toconvert the initiating ATG codon of hTRT into a Kozak consensus andcreate EcoRI and BglII sites for cloning. Also, oligo5′-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCC AGC-3′ (SEQ IDNO:614) is used to insert the IBI Flag (International BiotechnologiesInc. (IBI), Kodak, New Haven, Conn.) at the C-terminus and create EcoRIand BglII sites for cloning. Also, COD2866 is used to convert AmpS toAmpR and COD1941 is used to convert CatR to CatS.

[0915] pGRN129

[0916] The oligonucleotide5′-CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCT CACC-3′ (SEQID NO:609) was used by in vitro mutagenesis to convert Asp869 to an Alacodon (i.e. the second Asp of the DD motif was converted to an Alanineto create a dominant/negative hTRT mutant). This also created a MluIsite. Also, oligonucleotide 5′-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAG C-3′ SEQ IDNO:614) was used to insert the IBI Flag at the C-terminus and createEcoRI and BglII sites for cloning. Also, COD2866 was used to convertAmpS to AmpR and COD1941 was used to convert CatR to CatS.

[0917] pGRN130

[0918] The oligonucleotide 5′-CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCTCACC-3′ (SEQ ID NO:609) was used in in vitromutagenesis to convert the Asp869 codon into an Ala codon (i.e. thesecond Asp of the DD motif was converted to an Alanine to make adominant/negative variant protein). This also created an MluI site.Also, the oligonucleotide 5′-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3′ (SEQ ID NO:608) was used in in vitromutagenesis to convert the initiating ATG codon of the hTRT codingsequence into a Kozak consensus sequence and create EcoRI and BglIIsites for cloning. Also, COD2866 was used to convert AmpS to AmpR andCOD1941 was used to convert CatR.

[0919] pGRN131

[0920] The EcoRI fragment from pGRN128 containing the hTRT ORF withKozak sequence and IBI Flag mutations is inserted into the EcoRI site ofpBBS212 so that the hTRT ORF is expressed off the MPSV promoter. PlasmidpBSS212 contains a MPSV promoter, the CMV enhancer, and the SV40polyadenylation site.

[0921] pGRN132

[0922] The EcoRI fragment from pGRN128 containing the hTRT ORF withKozak sequence and 1131 Flag mutations is inserted into the EcoRI siteof pBBS212 so that the antisense of the hTRT ORF is expressed off theMPSV promoter.

[0923] pGRN133

[0924] The EcoRI fragment from pGRN121 containing the hTRT codingsequence was inserted into the EcoRI site of pBBS212 so that the hTRTprotein is expressed under the control of the MPSV promoter.

[0925] pGRN134

[0926] The EcoRI fragment from pGRN121 containing the hTRT codingsequence was inserted into the EcoRI site of pBBS212 so that theantisense of the hTRT coding sequence is expressed under the control ofthe MPSV promoter. The selectable markers used with this vector areChlor/HygB/PAC.

[0927] pGRN135

[0928] Plasmid pGRN126 was digested to completion with MscI and SmaI andreligated to delete over 95% of the hTRT coding sequence inserted. OneSmaI-MscI fragment was re-inserted during the process to recreate theCat activity for selection. This unpurified plasmid was then redigestedwith SalI and EcoRI and the fragment containing the initiating codon ofthe hTRT coding sequence was inserted into the SalI-EcoRI sites ofpBBS212. This makes an antisense expression plasmid expressing theantisense of the 5′UTR and 73 bases of the coding sequence. Theselectable markers used with this vector are Chlor/HygB/PAC.

[0929] pGRN136

[0930] The HindIII-SalI fragment from pGRN126 containing the hTRT codingsequence was inserted into the HindIII-SalI sites of pBBS242.

[0931] pGRN137

[0932] The SalI-Sse83871 fragment from pGRN130 containing the Kozaksequence was inserted into the SalI-Sse83871 sites of pGRN136.

[0933] pGRN138

[0934] The EcoRI fragment from pGRN124 containing hTRT minus the 3′UTRwas inserted into the EcoRI site of pEGFP-C2 such that the orientationof the hTRT is the same as the EGFP domain.

[0935] pGRN139

[0936] The oligonucleotide 5′-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAG C-3′ (SEQ IDNO:614) was used to insert the IBI Flag at the C-terminus of hTRT inpGRN125 and create EcoRI and BglII sites for cloning. Also, COD2866 wasused to convert AmpS to AmpR and COD1941 was used to convert CatR toCatS.

[0937] pGRN140

[0938] The NcoI fragment containing the upstream sequences of genomichTRT and the first intron of hTRT from lambdaG55 was inserted into theNcoI site of pBBS167. The fragment is oriented so that hTRT is in thesame direction as the Lac promoter.

[0939] pGRN141

[0940] The NcoI fragment containing the upstream sequences of genomichTRT and the first intron of hTRT from lambdaG55 was inserted into theNcoI site of pBBS167. The fragment is oriented so that hTRT is in theopposite direction as the Lac promoter.

[0941] pGRN142

[0942] The NotI fragment from lambdaGphi5 containing the complete ˜15kbp genomic insert including the hTRT gene promoter region was insertedin the NotI site of plasmid pBBS 185. The fragment is oriented so thatthe hTRT ORF is in the opposite orientation as the Lac promoter.

[0943] pGRN143

[0944] The NotI fragment from lambdaGphi5 containing the complete ˜15kbp genomic insert including the hTRT gene promoter region was insertedin the NotI site of plasmid pBBS 185. The fragment is oriented so thatthe hTRT ORF is in the same orientation as the Lac promoter.

[0945] pGRN144

[0946] SAL1 deletion of pGRN140 to remove lambda sequences.

[0947] pGRN145

[0948] This vector was constructed for the expression of hTRT sequencesin mammalian cells. The EcoRI fragment from pGRN137 containing the hTRTcoding sequence was inserted into the EcoRI site of pBBS212 to removethe portion of the sequence corresponding to the 5′UTR of hTRT mRNA. ThehTRT coding sequence is oriented so that it is expressed under thecontrol of the MPSV promoter. The selectable markers used with thisvector are Chlor/HygB/PAC.

[0949] pGRN146

[0950] This vector was constructed for the expression of hTRT sequencesin mammalian cells. The Sse83871-NotI fragment from pGRN130 containingthe D869A mutation of hTRT was inserted into the Sse83871-NotI sites ofpGRN137. The selectable markers used with this vector areAmpicillin/HygB/PAC.

[0951] pGRN147

[0952] The Sse83871-NotI fragment from pGRN139 containing the IBI Flagwas inserted into the Sse83871-NotI sites of pGRN137.

[0953] pGRN148

[0954] The BglII-Eco47III fragment from pGRN144 containing the promoterregion of hTRT was inserted into the BglII-NruI sites of pSEAP2 to makean hTRT promoter/reporter construct.

[0955] pGRN149

[0956] This vector is an intermediate vector for constructing a hTRTfusion protein expression vector. The mutagenic oligo5′-cttcaagaccatcctggactttcgaaacgcggccgccaccg cggtggagctcc-3′ (SEQ IDNO:615) was used to add a CSP45I site at the C-terminus of hTRT by invitro mutagenesis of pGRN125. The “stop” codon of hTRT was deleted andreplaced with a Csp45I site. The selectable marker that is used withthis vector is ampicillin.

[0957] pGRN150

[0958] The BglII-FspI fragment from pGRN144 containing the promoterregion of hTRT was inserted into the BglII-NruI sites of pSEAP2 to makean hTRT promoter/reporter construct.

[0959] pGRN151

[0960] This vector was constructed for the expression of hTRT sequencesin mammalian cells. The EcoRI fragment from pGRN147 containing the hTRTcoding sequence was inserted into the EcoRI site of pBBS212 to removethe portion of the sequence corresponding to the 5′UTR of the hTRT mRNA.The hTRT coding sequence is oriented so that it is expressed under thecontrol of the MPSV promoter. The selectable markers used with thisvector are Chlor/HygB/PAC.

[0961] pGRN152

[0962] The EcoRI fragment from pGRN146 containing the hTRT codingsequence was inserted into the EcoRI site of pBBS212 to remove theportion of the sequence corresponding to the 5′UTR of the hTRT. The hTRTcoding sequence is oriented so that it is expressed under the control ofthe MPSV promoter.

[0963] pGRN153

[0964] The StyI fragment from pGRN130 containing the D869—>A mutation ofhTRT (hTRT variant coding sequence) was inserted into the StyI sites ofpGRN158 to make a plasmid containing the hTRT coding sequence with aKozak consensus sequence at its 5′-end, an IBI FLAG sequence at its3′-end (the C-terminus encoding region), and the D869-->A mutation.

[0965] pGRN154

[0966] The EcoRI fragment of pGRN153 containing the hTRT gene wasinserted into the EcoRI site of plasmid pBS212 in an orientation suchthat the hTRT ORF is oriented in the same direction as the MPSVpromoter. This makes an MPSV-directed expression plasmid that expressesthe hTRT protein with a Kozak consensus sequence at its amino-terminalend, an 1131 FLAG at its carboxy-terminal end, and the D869-->A mutation

[0967] pGRN155

[0968] This vector was constructed for the expression of hTRT sequencesin mammalian cells. The insert included full length cDNA of hTRT minus5′ and 3′ UTR, and Kozak sequences. The EcoRI fragment from pGRN145containing the hTRT cDNA with the Kozak consensus and no 3′ or 5′ UTRwas inserted into the EcoRI site of p91023(B) such that the hTRT is inthe same orientation as the DHFR ORF. This makes a transient expressionvector for hTRT. The selectable marker used with this vector istetracycline.

[0969] pGRN156

[0970] This vector was constructed for the expression of hTRT sequencesin mammalian cells. The EcoRI fragment from pGRN146 containing the D869Amutation of the hTRT cDNA with the Kozak consensus and no 3′ or 5′ UTRwas inserted into the EcoRI site of p91023(B) such that the hTRT is inthe same orientation as the DHFR ORF. This makes a transient expressionvector for hTRT. The insert included full length cDNA of hTRT minus 5′and 3′ UTR, D869A, and Kozak sequences. The selectable marker used withthis vector is tetracycline.

[0971] pGRN157

[0972] This vector was constructed for the expression of hTRT sequencesin mammalian cells. The EcoRI fragment from pGRN147 containing the hTRTcDNA with the IBI FLAG at the C-terminus; the Kozak consensus and no 3′or 5′ UTR into the EcoRI site of p91023(B) such that the hTRT is in thesame orientation as the DHFR ORF. This makes a transient expressionvector for hTRT. The insert included full length cDNA of hTRT minus 5′and 3′ UTR, the IBI FLAG sequence, and Kozak sequences. The selectablemarker used with this vector is tetracycline.

[0973] pGRN158

[0974] This vector was constructed for the expression and mutagenesis ofTRT sequences in E. coli. The EcoRI fragment from pGRN151 containing thehTRT ORF was inserted into the EcoRI site of pBBS183 so that the hTRTORF is oriented in the opposite direction as the Lac promoter. Theinsert included full length cDNA of hTRT minus 5′ and 3′ UTR, IBI FLAGsequence, and Kozak sequences. The hTRT coding sequence is driven by aT7 promoter. The selectable marker used with this vector is amphicillin.

[0975] pGRN159

[0976] This vector was constructed for the expression and mutagenesis ofTRT sequences in E. coli. The NheI-Kpn1 fragment from pGRN138 containingthe EGFP to hTRT fusion was inserted into the XbaI-KpnI sites ofpBluescriptIIKS+. This makes a T7 expression vector for the fusionprotein (the coding sequence is driven by a T7 promoter). The insertincluded full length cDNA of hTRT minus the 3′ UTR as a fusion proteinwith EGFP. The selectable marker used with this vector is amphicillin.

[0977] pGRN160

[0978] This vector was constructed for the expression of antisense hTRsequences in mammalian cells. The coding sequence is operably linked toan MPSV promoter. The XhoI-NsiI fragment from pGRN90 containing the fulllength hTR ORF was inserted into the SalI-Sse8387I sites of pBBS295.This makes a transient/stable vector expressing hTR antisense RNA. A GPTmarker was incorporated into the vector. The selectable markers usedwith this vector are Chlor/gpt/PAC.

[0979] pGRN161

[0980] This vector was constructed for the expression of sense hTRsequences in mammalian cells. The XhoI-NniI fragment from pGRN89containing the full length hTR ORF was inserted into the SalI-Sse83871sites of pBBS295. This makes a transient/stable vector expressing hTR inthe sense orientation. The coding sequence is driven by an MPSVpromoter. A GPT marker was incorporated into the vector. The selectablemarkers used with this vector are Chlor/gpt/PAC.

[0981] pGRN162

[0982] The XhoI-NsiI fragment from pGRN87 containing the full length hTRORF was inserted into the SalI-Sse83871 sites of pBBS295. This makes atransient/stable vector expressing truncated hTR (from position +108 to+435) in the sense orientation.

[0983] pGRN163

[0984] This vector was constructed for the expression and mutagenesis ofTRT sequences in E. coli. The coding sequence is driven by a T7promoter. Oligonucleotide RA45 (5′-GCCACCCCCGCGCTGCCTCGAGCTCCCCGCTGC-3′;SEQ ID NO:616) is used in in vitro mutagenesis to change the initiatingmet in hTRT to Leu and introduce an XhoI site in the next two codonsafter the Leu. Also COD 1941 was used to change CatR to CatS, andintroduces a BSPH1 site, and COD 2866 was used to change AmpS to AmpR,introducing an FSP1 site. The selectable marker used with this vector isamphicillin.

[0985] pGRN164

[0986] This vector was constructed for the expression of hTR sequencesin E. coli. Primers hTR+1 5′-GGGGAAGCTTTAATACGACTCACTATAGGGTTGCGGAGGGTGGGCCTG-3′ (SEQ ID NO:617) and hTR+4455′-CCCCGGATCCTGCGCATGTGTGAGCCGAGTCCT GGG-3′ (SEQ ID NO:618) were used toamplify by PCR a fragment from pGRN33 containing the full length hTRwith the T7 promoter on the 5′ end (as in hTR+1). A BamHI-HindIII digestof the PCR product was put into the BamHI-HindIII sites of pUC119. Thecoding sequence operably linked to a T7 promoter. The selectable markerused with this vector is amphicillin. pGRN164 is also called phTR+1.

[0987] pGRN165

[0988] This vector was constructed for the expression and mutagenesis ofhTRT sequences in E. coli. The coding sequence is operably linked to aT7 promoter. The EcoRI fragment from pGRN145 containing the hTRT ORFwith a Kozak front end was inserted into the EcoRI site ofpBluescriptIISK+ so that the hTRT is oriented in the same direction asthe T7 promoter. The selectable marker used with this vector isamphicillin.

[0989] pGRN166

[0990] This vector was constructed for the expression and mutagenesis ofTRT sequences in mammalian cells. The coding sequence is operably linkedto a T7 promoter. The EcoRI fragment from pGRN151 containing the hTRTORF with a Kozak front end and IBI flag at the back end was insertedinto the EcoRI site of pBluescriptIISK+ so that the hTRT ORF is orientedin the same direction as the T7 promoter. The insert included fulllength cDNA of hTRT minus 5′ and 3′ UTR, FLAG sequence (Immunex Corp,Seattle Wash.), and Kozak sequences. The selectable marker used withthis vector is amphicillin.

[0991] pGRN167

[0992] AvRII-Stul fragment from pGRN144 containing the 5′ end of thehTRT ORF was inserted into the XbaI-StuI sites of pBBS161.

[0993] pGRN168

[0994] The EcoRI fragment from pGRN145 containing the optimized hTRTexpression cassette was inserted into the EcoRI site of pIND such thatthe hTRT coding sequence is in the same orientation as the miniCMVpromoter.

[0995] pGRN169

[0996] The EcoRI fragment from pGRN145 containing the optimized hTRTexpression cassette was inserted into the EcoRI site of pIND such thatthe hTRT is in the reverse orientation from the miniCMV promoter.

[0997] pGRN170

[0998] The EcoRI fragment from pGRN145 containing the optimized hTRTexpression cassette was inserted into the EcoRI site of pIND (sp 1) suchthat the hTRT is in the opposite orientation from the miniCMV promoter.

[0999] pGRN171

[1000] The Eco47III-NarI fragment from pGRN163 was inserted into theEco47III-NarI sites of pGRN167, putting the M 1 L mutation into afragment of the hTRT genomic DNA.

[1001] pGRN172

[1002] The BamHI-StuI fragment from pGRN171 containing the Met to Leumutation in the hTRT ORF was inserted into the BglII-NruI sites ofpSEAP2-Basic.

[1003] pGRN173

[1004] The EcoRV-ECO47III fragment from pGRN144 containing the 5′ end ofthe hTRT promoter region was inserted into the SrfI-Eco47III sites ofpGRN172. This makes a promoter reporter plasmid that contains thepromoter region of hTRT from approximately 2.3 kb upstream from thestart of the hTRT ORF to just after the first intron in the codingregion, with the Met1-->Leu mutation.

[1005] pGRN174

[1006] The EcoRI fragment from pGRN145 containing the “optimized” hTRTexpression cassette was inserted into the EcoRI site of pIND (sp1) suchthat the hTRT is in the same orientation as the miniCMV promoter.

Example 7 Reconstitution of Telomerase Activity

[1007] A. Co-Expression of hTRT and hTR In Vitro

[1008] In this example, the coexpression of hTRT and hTR using an invitro cell-free expression system is described. These resultsdemonstrate that the hTRT polypeptide encoded by pGRN121 encodes acatalytically active telomerase protein and that in vitro reconstitution(IVR) of the telomerase RNP can be accomplished using recombinantlyexpressed hTRT and hTR.

[1009] Telomerase activity was reconstituted by adding linearizedplasmids of hTRT (PGRN121; 1 μg DNA digested with Xba I) and hTR(phTR+1; 1 μg digested with FspI) to a coupled transcription-translationreticulocyte lysate system (Promega TNT™). phTR+1 is a plasmid which,when linearized with FspI and then transcribed by T7 RNA polymerase,generates a 445 nucleotide transcript beginning with nucleotide +1 andextending to nucleotide 446 of hTR (Autexier et al., 1996, EMBO J.15:5928). For a 50 μl reaction the following components were added: 2 μlTNT™ buffer, 1 μl TNT T7 RNA polymerase, 1 μl 1 mM amino acid mixture,40 units Rnasin™ RNase inhibitor, 1 μg each linearized template DNA, and25 μl TNT™ reticulocyte lysate. Components were added in the ratiorecommended by the manufacturer and were incubated for 90 min at 30° C.Transcription was under the direction of the T7 promoter and could alsobe carried out prior to the addition of reticulocyte lysate with similarresults. After incubation, 5 and 10 μl of the programmedtranscription-translation reaction were assayed for telomerase activityby TRAP as previously described (Autexier et al., supra) using 20 cyclesof PCR to amplify the signal.

[1010] The results of the reconstitution are shown in FIG. 10. For eachtranscription/translation reaction assayed there are 3 lanes: The first2 lanes are duplicate assays and the third lane is a duplicate sampleheat denatured (95° C., 5 min) prior to the TRAP phase to rule out PCRgenerated artifacts.

[1011] As shown in FIG. 10, reticulocyte lysate alone has no detectabletelomerase activity (lane 6). Similarly, no detectable activity isobserved when either hTR alone (lane 1) or full length hTRT gene (lane4) are added to the lysate. When both components are added (lane 2),telomerase activity is generated as demonstrated by the characteristicrepeat ladder pattern. When the carboxyl-terminal region of the hTRTgene is removed by digestion of the vector with NcoI (“truncated hTRT”)telomerase activity is abolished (lane 3). Lane 5 shows that translationof the truncated hTRT alone does not generate telomerase activity. Lane“R8” shows a positive control for a telomerase product ladder generatedby TRAP of TSR8, a synthetic telomerase product having a nucleotidesequence of 5′-ATTCCGTCGAGCAGAGTTAG[GGTTAG]₇-3′ (SEQ ID NO:619).

[1012] It was also observed that purification of IVR telomerase resultedin a stronger signal and/or reduced background in certain telomeraseactivity assays. In some experiments, IVR telomerase activity fromco-synthesized components was enriched by fractionation of TNT reactionsover DEAE anion exchange membranes (Millipore Ultrafree-MC): 200 μl ofthe hTRT/hTR TNT reaction was passed through a single DEAE membrane. Themembrane was washed with 400 μl of 0.2 M NaCl in buffer A (20 mMHEPES-KOH pH 7.9, 2 mM MgCl₂, 1 mM EGTA, 10% glycerol, 0.1% NonidetP-40, 0.1 mM phenylmethylsulfonyl fluoride) and IVR telomerase waseluted from the membrane with 80 μl of 1 M NaCl in buffer A.Alternatively, batch chromatography was used: 400 μl of the TNT reactionwas partially purified by batch chromatography using 25 μl of Toso-HaasQ-650M resin. After binding telomerase to the resin, it was washed with0.1 M NaCl in buffer A, followed by a second wash with 0.18 M NaCl inbuffer A and eluted with 100 μl of 0.3 M NaCl in buffer A.

[1013] B. Mixing of hTRT and hTR In Vitro

[1014] In vitro reconstitution of telomerase activity was alsoaccomplished by mixing. hTRT was transcribed and translated as describedsupra, but without the addition of the hTR plasmid. Reconstitution ofthe telomerase RNP was then accomplished by mixing the hTRT translationmixture with hTR (previously generated by T7 RNA polymerasetranscription from phTR+1−Fsp) in the ratio of 2 μl of hTRT translationmix to 2 μL of hTR (1 ug) then incubated for 90 minutes at 30° C. Thereaction conditions were adjusted to a KCl concentration of about 0.2 M.(The presence of KCl at a concentration of about 0.1 M to about 1.0 Mmay enhance telomerase activity or telomerase reconstitution in IVR).This method of hTRT/hTR reconstitution is referred to as “linkedreconstitution” or “linked IVR.” Telomerase activity is present (i.e.,can be detected) in this mixture. Improved signal was observed followingpartial purification of the activity by DEAE chromatography. In thiscase Millipore Ultrafree-MC DEAE Centrifugal Filter Devices were usedaccording to the manufacturer's directions). The buffers used werehypo0.1, hypo0.2, and hypo1.0, where hypo is 20 mM Hepes-KOH, pH 7.9, 2mM MgCl₂, 1 mM EGTA, 10% glycerol, 0.1% NP-40, 1 mM DTT, 1 mMNa-metabisulfite, 1 mM benzamidine, and 0.2 mMphenylmethylsulfonylflouride (PMSF), and where 0.1, 0.2 and 1.0 refersto 0.1, 0.2 or 1.0 M KCL. The filters were pre-conditioned with hypo1.0,washed with hypo0.1, the reconstituted telomerase was loaded, the columnwas washed with hypo0.1 then hypo0.2, and the reconstituted telomerasewas eluted with hypo1.0 at half the volume as was loaded. Thisformulation could be stored frozen at −70° C. and retains activity.

[1015] Telomerase activity was assayed in a two step procedure. In stepone, a conventional telomerase assay was performed as described inMorin, 1989, Cell 59: 521, except no radiolabel was used. In step two,an aliquot was assayed by the TRAP procedure for 20-30 cycles asdescribed supra. The conventional assay was performed by assaying 1-10μl of reconstituted telomerase in 40-50 μl final volume of 25 mMTris-HCl, pH 8.3, 50 mM K-acetate, 1 mM EGTA, 1 mM MgCl₂, 2 mM DATP, 2mM TTP, 10 uM dGTP, and 1 uM primer (usually M2,5′-AATCCGTCGAGCAGAGTT;SEQ ID NO:620) at 30° C. for 60-180 minutes. The reaction was stopped byheating to 95° C. for 5 minutes and 1-10 μl of the first step mixturewas carried onto the step two TRAP reaction (50 ul).

[1016] In additional experiments, the synthesis of hTRT and hTR duringin vitro reconstitution was monitored by ³⁵S-methionine incorporationand Northern blotting, respectively. Proteins of approximately thepredicted size were synthesized for hTRT (127 kD), hTRT-Nco (85 kD), andpro90hTRT (90 kD) in approximately equal molar amounts relative to eachother. The Northern analysis indicated hTR synthesis was the correctsize (445 nucleotides) and predominantly intact.

[1017] High levels of reconstitution and telomerase activity were alsoobtained with 2 μg of linearized pGRN121 in a 50 μl TnT reaction asdescribed supra (Example 7A) except that in place of the hTR template, 4pmol (0.6 μg) of hTR RNA (previously generated by T7 RNA polymerasetranscription from phTR+1−Fsp) was added at the beginning of the TnTreaction and the reaction was incubated at 30EC for 90-120 minutes.Slightly greater (2-5 times) activity was achieved using 1 μg ofsupercoiled XhTRT- and 16 pmol (2.4 μg) of pre-synthesized hTR RNA setup in a 50 μl TnT reaction, as described supra, with incubation at 30ECfor 90-120 minutes. XhTRT-E is an hTRT construct in the pcDNA3.1/HisXpress vector (Invitrogen) in which an optimized ribosome recognitionsite (Kozak consensus), six histidine residues, and an epitope tag arefused with the hTRT open reading frame.

[1018] Variations of the reconstitution protocols, supra, will beapparent to those of skill. For example, the time and temperature ofreconstitution, and presence or concentration of components such asmonovalent salt (e.g. NaCl, KCl, potassium acetate, potassium glutamate,and the like), divalent salt (MgCl₂, MnCl₂, MgSO₄, and the like),denaturants (urea, formamide, and the like), detergents (NP-40, Tween,CHAPS, and the like), and alternative improved purification procedures(such as immunoprecipitation, affinity or standard chromatography) canbe employed. These and other parameters can be varied in a systematicway to optimize conditions for particular assays or other reconstitutionprotocols.

[1019] C. Reconstitution Using hTRT Variants and Fusion Proteins

[1020] Reconstitution of telomerase catalytic activity occurred whenEGFP-hTRT, a fusion of the enhanced green fluorescent protein to hTRT(see Examples 6 and 15), or epitope-tagged hTRT (IBI FLAG, see Example6) both reconstituted telomerase activity at approximately wild-typelevels were coexpressed with hTR.

[1021] In contrast, telomerase activity was not reconstituted when avariant hTRT, pro90hTRT (missing RT motifs B′, C, D, and E) was used.This demonstrates that pro90hTRT does not possess full telomerasecatalytic activity, although it may have other partial activities (e.g.,RNA [i.e. hTR] binding ability and function as dominant-negativeregulator of telomerase in vivo as described supra).

[1022] D. Assay of In Vitro Reconstituted Telomerase Activity Using theGel Blot and Conventional Telomerase Assay

[1023] The following example demonstrates that in vitro reconstituted(IVR) telomerase can be assayed using conventional telomerase assays inaddition to amplification-based assays (i.e., TRAP). IVR telomerase asdescribed in part (B), supra (the “linked reconstitution method”)followed by DEAE purification, as described supra was assayed using thegel blot assay using the following reaction conditions; 1-10 μl oflinked IVR telomerase in 40 μl final volume of 25 mM Tris-HCl, pH 8.3,50 mM K-acetate, 1 mM EGTA, 1 mM MgCl₂, 0.8 mM dATP, 0.8 mM TTP, 1.0 mMdGTP, and 1 uM primer (M2, supra; or H3.03, 5′-TTAGGGTTAGGGTTAGGG; SEQID NO:621) at 30° C. for 180 minutes. The telomeric DNA synthesized wasisolated by standard procedures, separated on a 8% polyacrylamide, 8 Murea gel, transferred to a nylon membrane, and probed using the³²P-(CCCTAA)n riboprobe used in the dot-blot assay. The probe identifieda six nucleotide ladder in the lane representing 10 μl of IVR telomerasethat was equivalent to the ladder observed for 5 μl of native nucleartelomerase purified by mono Q and heparin chromatography. The resultsshow that IVR telomerase possesses processive telomerase catalyticactivity equivalent to native telomerase.

[1024] Linked IVR telomerase was also assayed by the conventional³²P-dGTP incorporation telomerase assay. IVR telomerase prepared by thelinked reconstitution method followed by DEAE purification, as describedabove, was assayed under both processive and non-processive reactionconditions. Assay conditions were 5-10 μl of linked VR telomerase in 40μl final volume of 25 mM Tris-HCl, pH 8.3, 50 mM K-acetate, 1 mM EGTA, 1mM MgCl₂, 2 mM dATP, 2 mM TTP, with 10 uM ³²P-dGTP (72 Ci/mmol) [forassay of processive conditions] or 1 uM ³²P-dGTP (720 Ci/mmol) [fornon-processive], and 1 uM primer (i.e., H3.03, supra) at 30° C. [for theprocessive reaction] or 37° C. [for the non-processive reaction] for 180minutes. The telomeric DNA synthesized was isolated by standardprocedures and separated on a 8% polyacrylamide, 8 M urea gel sequencinggel. The processive reaction showed a weak six nucleotide ladderconsistent with a processive telomerase reaction, and the non-processivereaction added one repeat, a pattern equivalent to a control reactionwith a native telomerase preparation. Conventional assays using IVRtelomerase are useful in screens for telomerase modulators, as describedherein, as well as other uses such as elucidation of the structural andfunctional properties of telomerase.

[1025] E. In Vitro Reconstituted Telomerase Recognizes Primer 3′ Termini

[1026] This experiment demonstrates that IVR telomerase recognizesprimer 3′ termini equivalently to native (purified) telomerase.Telomerase forms a base-paired duplex between the primer 3′ end and thetemplate region of hTR and adds the next specified nucleotide (Morin,1989, supra). To verify that IVR (recombinant) telomerase has the sameproperty, the reactions of primers with ---GGG or ---TAG 3′ termini(AATCCGTCGAGCAGAGGG; SEQ ID NO:622 and AATCCGTCGAGCAGATAG; SEQ IDNO:623) were compared to a primer having a ---GTT 3′ terminus (M2 supra)using IVR and native telomerase assayed by the two stepconventional/TRAP assay detailed above. The product ladders of the---GGG and ---TAG primers were shifted +4 and +2, respectively, whencompared to the standard primer (---GTT 3′ end), the same effect as wasobserved with native telomerase. This experiment demonstrates IVR andnative telomerases recognize primer termini in a similar manner.

[1027] These results (along with the results supra showing that IVRtelomerase possesses both processive and non-processive catalyticactivity) indicate that IVR telomerase has similar structure andproperties compared to native or purified telomerase.

Example 8 Production of Anti-hTRT Antibodies

[1028] A. Production of Anti-hTRT Antibodies Against hTRT Peptides

[1029] To produce anti-hTRT antibodies, the following peptides from hTRTwere synthesized with the addition of C (cysteine) as the amino terminalresidue (see FIG. 54). S-1: FFY VTE TTF QKN RLF FYR KSV WSK SEQ IDNO:232 S-2: RQH LKR VQL RDV SEA EVR QHR EA SEQ ID NO:233 S-3: ART FRREKR AER LTS RVK ALF SVL NYE SEQ ID NO:234 A-3: PAL LTS RLR FIP KPD GLRPIV NMD YVV SEQ ID NO:237

[1030] The cysteine moiety was used to immobilize (i.e., covalentlylink) the peptides to BSA and KLH [keyhole limpet hemocyanin] carrierproteins. The KLH-peptides were used as antigen. The BSA-peptideconjugates served as material for ELISAs for testing the specificity ofimmune antisera.

[1031] The KLH-peptide conjugates were injected into New Zealand Whiterabbits. The initial injections are made by placing the injectantproximal to the axillary and inguinal lymph nodes. Subsequent injectionswere made intramuscularly. For initial injections, the antigen wasemulsified with Freund's complete adjuvant; for subsequent injections,Freund's incomplete adjuvant was used. Rabbits follow a three week boostcycle, in which 50 ml of blood yielding 20-25 ml of serum is taken 10days after each boost. Antisera against each of the four peptidesrecognized the hTRT moiety of recombinant hTRT fusion protein(GST-HIS₈-hTRT-fragment 2426 to 3274); see Example 6) on western blots.

[1032] Using a partially purified telomerase fraction from human 293cells (approximately 1000-fold purification compared to a crude nuclearextract) that was produced as described in PCT application No. 97/06012and affinity purified anti-S-2 antibodies, a 130 kd protein doubletcould be detected on a western blot. A sensitive chemiluminescencedetection method was employed (SuperSignal chemiluminescence substrates,Pierce) but the signal on the blot was weak, suggesting that hTRT ispresent in low or very low abundance in these immortal cells. Theobservation of a doublet is consistent with a post-translationalmodification of hTRT, i.e., phosphorylation or glycosylation.

[1033] For affinity purification, the S-2 peptide was immobilized toSulfoLink (Pierce, Rockford Ill.) through its N-terminal Cysteineresidue according to the manufacturer's protocol. First bleed serum froma rabbit immunized with the KLH-S-2 peptide antigen was loaded over athe S-2-SulfoLink and antibodies specifically bound to the S-2 peptidewere eluted.

[1034] B. Production of Anti-hTRT Antibodies Against hTRT FusionProteins

[1035] GST-hTRT fusion proteins were expressed in E. coli as theGST-hTRT fragment #4 (nucleotides 3272-4177) and the GST-HIS8-hTRTfragment #3 (nucleotides 2426 to 3274) proteins described in Example 6.The fusion proteins were purified as insoluble protein, and the purityof the antigens was assayed by SDS polyacrylamide gels and estimated tobe about 75% pure for the GST-hTRT fragment #4 recombinant protein andmore than 75% pure for GST-HIS8-hTRT fragment #3 recombinant protein.Routine methods may be used to obtain these and other fusion proteins ata purity of greater than 90%. These recombinant proteins were used toimmunize both rabbits and mice, as described above.

[1036] The first and second bleeds from both the mice and rabbits weretested for the presence of anti-hTRT antibodies after removal ofanti-GST antibodies using a matrix containing immobilized GST. Theantisera were tested for anti-hTRT antibodies by Western blotting usingimmobilized recombinant GST-hTRT fusion protein, and byimmunoprecipitation using partially purified native telomerase enzyme.While no signal was observed in these early bleeds, titers of anti-hTRTantibodies, as expected, increased in subsequent bleeds.

Example 9 Detection of an hTRT mRNA Corresponding to Δ182 RNA Variant

[1037] Poly A⁺ RNA from human testis and the 293 cell line was analyzedfor hTRT mRNA using RT-PCR and nested primers. The first primer set wasTCP 1.1 and TCP 1.15; the second primer set was TCP 1.14 and BTCP6.Amplification from each gave two products differing by 182 bp; thelarger and smaller products from testis RNA were sequenced and found tocorrespond exactly to pGRN121(FIG. 16) and the 712562 clone (FIG. 18),respectively. The variant hTRT RNA product has been observed in mRNAfrom SW39i, OVCAR4, 293, and Testes.

[1038] Additional experiments were carried out to demonstrate that theAl 82 cDNA was not an artifact of reverse transcription. Briefly,full-length hTRT RNA (i.e., without the deletion) was produced by invitro transcription of pGRN121 for use as a template for RT-PCR.Separate cDNA synthesis reactions were carried out using Superscript7reverse transcriptase (Bethesda Research Laboratories, Bethesda Md.) at42° or 50° C., and with random-primers or a specific primer. After 15PCR cycles the longer product was detectable; however, the smallerproduct (i.e., corresponding to the deletion) was not detectable evenafter 30 or more cycles. This indicates that the RT-PCR product is notan artifact.

Example 10 Sequencing of Testis hTRT mRNA

[1039] The sequence of the testis form of hTRT RNA was determined bydirect manual sequencing of DNA fragments generated by PCR from testiscDNA (Marathon Testes cDNA, Clontech, San Diego Calif.) using aThermoSequenase radiolabeled terminator cycle sequencing kit (AmershamLife Science). The PCR step was performed by a nested PCR, as shown inTable 8. In all cases a negative control reaction with primers but nocDNA was performed. The absence of product in the control reactiondemonstrated that the products derived from the reaction with cDNApresent were not due to contamination of hTRT from pGRN121 or other cellsources (e.g., 293 cells). The DNA fragments were excised from agarosegels to purify the DNA prior to sequencing.

[1040] The testis mRNA sequence corresponding to bases 27 to 3553 of thepGRN121 insert sequence, and containing the entire hTRT ORF (bases 56 to3451) was obtained. There were no differences between the testis and thepGRN121 sequences in this region. TABLE 8 Primer Primer Final FragmentSet 1 Set 2 Size Primers for Seq OA na K320/K322 208 K320, K322 A K320/TCP1.40/ 556 TCP1.52, TCP1.39, TCP1.43 TCP1.34 K322, TCP1.40, TCP1.41,TCP1.30, TCP1.34, TCP1.49 B TCP1.42/ TCP1.35/ 492 TCP1.35, TCP1.28,TCP1.32B TCP1.21 TCP1.38, TCP1.21, TCP1.46, TCP1.33, TCP1.48 C TCP1.65/TCP1.67/ 818 TCP1.67, TCP1.32, TCP1.66 TCP1.68 TCP1.69, TCP1.68,TCP1.24, TCP1.44, K303 D2 K304/ Lt1/ 546 Lt2, Lt1, TCP1.6, billTCP6TCP1.6 bi11TCP4, TCP1.13, TCP1.77, TCP1.1 D3 TCP1.12/ TCP1.14/ 604TCP1.6, TCP1.14, TCP1.7 TCP1.15 TCP1.73, TCP1.78, TCP1.25, TCP1.15,TCP1.76 EF na TCP1.74/ 201 TCP1.74, TCP1.7, TCP1.7 TCP1.75, TCP1.15,TCP1.3 E TCP1.3/ TCP1.2/ 687 TCP1.2, TCP1.8, TCP1.4 TCP1.9 TCP1.9,TCP1.26 F TCP1.26/ TCP1.10 377 TCP1.4, TCP1.10, UTR2 TCP1.4 TCP1.11

Example 11 Detection of hTRT mRNA by RNase Protection

[1041] RNase protection assays can be used to detect, monitor, ordiagnose the presence of an hTRT mRNA or variant mRNA. One illustrativeRNAse protection probe is an in vitro synthesized RNA comprised ofsequences complementary to hTRT mRNA sequences and additional,non-complementary sequences. The latter sequences are included todistinguish the full-length probe from the fragment of the probe thatresults from a positive result in the assay: in a positive assay, thecomplementary sequences of the probe are protected from RNase digestion,because they are hybridized to hTRT mRNA. The non-complementarysequences are digested away from the probe in the presence of RNase andtarget complementary nucleic acid.

[1042] Two RNAse protection probes are described for illustrativepurposes; either can be used in the assay. The probes differ in theirsequences complementary to hTRT, but contain identical non-complementarysequences, in this embodiment, derived from the SV40 late mRNA leadersequence. From 5′-3′, one probe is comprised of 33 nucleotides ofnon-complementary sequence and 194 nucleotides of sequence complementaryto hTRT nucleotides 2513-2707 for a full length probe size of 227nucleotides. From 5′-3′, the second probe is comprised of 33 nucleotidesof non-complementary sequence and 198 nucleotides of sequencecomplementary to hTRT nucleotides 2837-3035 for a full length probe sizeof 231 nucleotides. To conduct the assay, either probe can be hybridizedto RNA, i.e., polyA+ RNA, from a test sample, and Ti ribonuclease andRNase A are then added. After digestion, probe RNA is purified andanalyzed by gel electrophoresis. Detection of a 194 nucleotide fragmentof the 227 nucleotide probe or a 198 nucleotide fragment of the 231nucleotide probe is indicative of hTRT mRNA in the sample.

[1043] The illustrative RNAse protection probes described in thisexample can be generated by in vitro transcription using T7 RNApolymerase. Radioactive or otherwise labeled ribonucleotides can beincluded for synthesis of labeled probes. The templates for the in vitrotranscription reaction to produce the RNA probes are PCR products. Theseillustrative probes can be synthesized using T7 polymerase following PCRamplification of pGRN121 DNA using primers that span the correspondingcomplementary region of the hTRT gene or mRNA. In addition, thedownstream primer contains T7 RNA polymerase promoter sequences and thenon-complementary sequences.

[1044] For generation of the first RNAse protection probe, the PCRproduct from the following primer pair (T701 and reverse01) is used:T701 5′-GGGAGATCT TAATACGACTCACTATAG (SEQ ID NO:624) ATTCA GGCCATGGTGCTGCGCCGGC TGTCA GGCTCCC ACGACGTAGT CCATGTTCAC-3′; and reverse015′-GGGTCTAGAT CCGGAAGAGTGT (SEQ ID NO:625) CTGGAGCAAG-3′.

[1045] For generation of the second RNase protection probe, the PCRproduct from the following primer pair (T702 and reverse02) is used:T702 5′-GGGAGATCT TAATACGACTCACTATAG (SEQ ID NO:626) ATTCA GGCCATGGTGCTGCGCCGGC TGTCA GGGCG GCCTTCTGGA CCACGGCATA CC-3′; and reverse02 5′-GGTCTAGA CGATATCC ACAGGGCCTG (SEQ ID NO:672) GCGC-3′.

Example 12 Construction of a Phylogenetic Tree Comparing hTRT and OtherReverse Transcriptases

[1046] A phylogenetic tree (FIG. 6) was constructed by comparison of theseven RT domains defined by Xiong and Eickbush (1990, EMBO J. 9:3353).After sequence alignment of motifs 1, 2, and A-E from 4 TRTs, 67 RTs,and 3 RNA polymerases, the tree was constructed using the NJ (NeighborJoining) method (Saitou and Nei, 1987, Mol. Biol. Evol. 4:406). Elementsfrom the same class that are located on the same branch of the tree aresimplified as a box. The length of each box corresponds to the mostdivergent element within that box.

[1047] The TRTs appear to be more closely related to RTs associated withmsDNA, group II introns, and non-LTR (Long Terminal Repeat)retrotransposons than to the LTR-retrotransposon and viral RTs. Therelationship of the telomerase RTs to the non-LTR branch ofretroelements is intriguing, given that these latter elements havereplaced telomerase for telomere maintenance in Drosophila. However, themost striking finding is that the TRTs form a discrete subgroup, almostas closely related to the RNA-dependent RNA polymerases of plus-strandedRNA viruses such as poliovirus as to any of the previously known RTs.Considering that the four telomerase genes come from evolutionarilydistant organisms—protozoan, fungi, and mammal—this separate groupingcannot be explained by lack of phylogenetic diversity in the data set.Instead, this deep bifurcation suggests that the telomerase RTs are anancient group, perhaps originating with the first eukaryote.

[1048] GenBank protein identification or accession numbers used in thephylogenetic analysis were: msDNAs (94535, 134069, 134074, 134075,134078), group II introns (483039, 101880, 1332208, 1334433, 1334435,133345, 1353081), mitochondrial plasmid/RTL (903835, 134084), non-LTRretrotransposons (140023, 84806, 103221, 103353, 134083, 435415, 103015,1335673, 85020, 141475, 106903, 130402, U0551, 903695, 940390, 2055276,L08889), LTR retrotransposons (74599, 85105, 130582, 99712, 83589,84126, 479443, 224319, 130398, 130583, 1335652, 173088, 226407, 101042,1078824), hepadnaviruses (I 18876, 1706510, 118894), caulimoviruses(331554, 130600, 130593, 93553), retroviruses (130601, 325465, 74601,130587, 130671, 130607, 130629, 130589, 130631, 1346746, 130651, 130635,1780973, 130646). Alignment was analyzed using ClustalW 1.5 [J. D.Thompson, D. G. Higgins, T. J. Gibson, Nucleic Acids Res. 22, 4673(1994)] and PHYLIP 3.5 [J. Felsenstein, Cladisfics 5, 164 (1989)].

Example 13 Transfection of Cultured Human Fibroblasts (BJ) with ControlPlasmid and Plasmid Encoding hTRT

[1049] This example demonstrates that expression of recombinant hTRTprotein in a mammalian cell results in the generation of an activetelomerase.

[1050] Subconfluent BJ fibroblasts were trypsinized and resuspended infresh medium (DMEM/199 containing 10% Fetal Calf Serum) at aconcentration of 4×10⁶ cells/ml. The cells were transfected usingelectroporation with the BioRad Gene Pulser™ electroporator. Optionally,one may also transfect cells using Superfect™ reagent (Qiagen) inaccordance with the manufacturer's instructions. For electroporation,500 μl of the cell suspension were placed in an electroporation cuvette(BioRad, 0.4 cm electrode gap). Plasmid DNA (2 μg) was added to thecuvettes and the suspension was gently mixed and incubated on ice for 5minutes. The control plasmid (pBBS212) contained no insert behind theMPSV promoter and the experimental plasmid (pGRN133) expressed hTRT fromthe MPSV promoter. The cells were electroporated at 300 Volts and 960μFD. After the pulse was delivered, the cuvettes were placed on ice forapproximately 5 minutes prior to plating on 100 mm tissue culture dishesin medium. After 6 hours, the medium was replaced with fresh medium. 72hours after the transfection, the cells were trypsinized, washed oncewith PBS, pelleted and stored frozen at −80° C. Cell extracts wereprepared at a concentration of 25,000 cells/μl by a modified detergentlysis method (see Bodnar et al., 1996, Exp. Cell Res. 228:58; Kim etal., 1994, Science 266:2011, and as described in patents andpublications relating to the TRAP assay, supra) and telomerase activityin the cell extracts was determined using a modified PCR-based TRAPassay (Kim et al., 1994, Bodnar et al., 1996). Briefly, 5×10⁴ cellequivalents were used in the telomerase primer extension portion of thereaction. While the extract is typically taken directly from thetelomerase extension reaction to the PCR amplification, one may alsoextract once with phenol/chloroform and once with chloroform prior tothe PCR amplification. One-fifth of the material was used in the PCRamplification portion of the TRAP reaction (approximately 10,000 cellequivalents). One half of the TRAP reaction was loaded onto the gel foranalysis, such that each lane in FIG. 25 represents reaction productsfrom 5,000 cell equivalents. Extracts from cells transfected withpGRN133 were positive for telomerase activity while extracts fromuntransfected (not shown) or control plasmid transfected cells showed notelomerase activity. Similar experiments using RPE cells gave the sameresult.

[1051] Reconstitution in BJ cells was also carried out using other hTRTconstructs (i.e., pGRN145, pGRN155 and pGRN138). Reconstitution usingthese constructs appeared to result in more telomerase activity than inthe pGRN133 transfected cells.

[1052] The highest level of telomerase activity was achieved usingpGRN155. As discussed supra, pGRN155 is a vector containing theadenovirus major late promoter as a controlling element for theexpression of hTRT and was shown to reconstitute telomerase activitywhen transfected into BJ cells.

[1053] Notably, when reconstitution using the hTRT-GFP fusion proteinpGRN138 (which localizes to the nucleus, see Example 15, infra) wasperformed either in vitro (see Example 7) or in vivo (transfection intoBJ cells) telomerase activity resulted. By transfection into BJ cells,for example, as described supra, telomerase activity was comparable tothat resulting from reconstitution in vitro using pGRN133 or pGRN145.

[1054] Similar results were obtained upon transfection of normal humanretinal pigmented epithelial (RPE) with the hTRT expression vectors ofthe invention. The senescence of RPE cells is believed to contribute toor cause the disease of age-related macular degeneration. RPE cellstreated in accordance with the methods of the invention using the hTRTexpression vectors of the invention should exhibit delayed senescence,as compared to untreated cells, and so be useful in transplantationtherapies to treat or prevent age-related macular degeneration.

Example 14 Promoter Reporter Construct

[1055] This example describes the construction of plasmids in whichreporter genes are operably linked to hTRT upstream sequences containingpromoter elements. The vectors have numerous uses, includingidentification of cis and trans transcriptional regulatory factors invivo and for screening of agents capable of modulating (e.g., activatingor inhibiting) hTRT expression (e.g., drug screening). Although a numberof reporters may be used (e.g., firefly luciferase, β-glucuronidase,β-galactosidase, chloramphenicol acetyl transferase, and GFP and thelike), the human secreted alkaline phosphatase (SEAP; CloneTech) wasused for initial experiments. The SEAP reporter gene encodes a truncatedform of the placental enzyme which lacks the membrane anchoring domain,thereby allowing the protein to be secreted efficiently from transfectedcells. Levels of SEAP activity detected in the culture medium have beenshown to be directly proportional to changes in intracellularconcentrations of SEAP mRNA and protein (Berger et al., 1988, Gene 66:1;Cullen et al., 1992, Meth. Enzymol. 216:362).

[1056] Four constructs (pGRN148, pGRN150, “pSEAP2 basic” (no promotersequences=negative control) and “pSEAP2 control” (contains the SV40early promoter and enhancer) were transfected in triplicate into mortaland immortal cells.

[1057] Plasmid pGRN148 was constructed as illustrated in FIG. 9.Briefly, a Bgl2-Eco47III fragment from pGRN144 was digested and clonedinto the BglII-NruI site of pSeap2Basic (Clontech, San Diego, Calif.). Asecond reporter-promoter, plasmid pGRN150, includes sequences from thehTRT intron described in Example 3, to employ regulatory sequences thatmay be present in the intron. The initiating Met is mutated to Leu, sothat the second ATG following the promoter region will be the initiatingATG of the SEAP ORF.

[1058] The pGRN148 and pGRN150 constructs (which include the hTRTpromoter) were transfected into mortal (BJ cells) and immortal (293)cells. All transfections were done in parallel with two controlplasmids: one negative control plasmid (pSEAP basic) and one positivecontrol plasmid (pSEAP control which contains the SV40 early promoterand the SV40 enhancer).

[1059] In immortal cells, pGRN148 and pGRN150 constructs appear to driveSEAP expression as efficiently as the pSEAP2 positive control(containing the SV40 early promoter and enhancer). In contrast, inmortal cells only the pSEAP2 control gave detectable activity. Theseresults indicate that, as expected, hTRT promoter sequences are activein tumor cells but not in mortal cells.

[1060] Similar results were obtained using another normal cell line(RPE, or retinal pigmental epithelial cells). In RPE cells transfectedwith pGRN150 (containing 2.2 KB of upstream genomic sequence), the hTRTpromoter region was inactive while the pSEAP2 control plasmid wasactive.

[1061] As noted supra, plasmids in which reporter genes are operablylinked to hTRT upstream sequences containing promoter elements areextremely useful for identification and screening of telomerase activitymodulatory agents, using both transient and stable transfectiontechniques. In one approach, for example, stable transformants ofpGRN148 are made in telomerase negative and telomerase positive cells bycotransfection with a eukaryotic selectable marker (such as neo)according to Ausubel et al., 1997, supra. The resulting cell lines areused for screening of putative telomerase modulatory agents, forexample, by comparing hTRT-promoter-driven expression in the presenceand absence of a test compound.

[1062] The promoter-reporter (and other) vectors of the invention arealso used to identify trans- and cis-acting transcriptional andtranslational regulatory elements. Examples of cis-actingtranscriptional regulatory elements include promoters and enhancers ofthe telomerase gene. The identification and isolation of cis- andtrans-acting regulatory agents provide for further methods and reagentsfor identifying agents that modulate transcription and translation oftelomerase.

[1063] To identify sequences or elements that play a role in hTRTexpression, expression was tested using promoter-reporter constructswith varying amounts of the upstream region (5′ to the transcriptioninitiation site) of the hTRT gene. Experiments were conducted using pGRN150 [which contains approximately 2405 bp of genomic sequence upstreamof the most 5′ nucleotide present in the hTRT cDNA], pGRN 176 [whichcontains approximately 186 bp of genomic sequence upstream of the most5′ nucleotide present in the hTRT cDNA] and pGRN 175 [which containsapproximately 77 bp of genomic sequence upstream of the most 5′nucleotide present in the hTRT cDNA]. The following sequence is presentin pGRN 176 but not pGRN 175: 5′-GTGGCGGAGGGACTGGGGACCCGGGCACCGGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCGTCCGCGCGGAACCCCGCCCCGTCCCGAACCCTTCCCGGGTCCCCGGCCCAGCCCCTTCCGGG-3′ (SEQ ID NO:726).

[1064] When transfected into mortal cells (RPE and BJ), the pGRN 175promoter was active, while the pGRN 176 and pGRN 150 promoters were notactive. These results demonstrate that the approximately 120 basepairregion present in pGRN 176 but not pGRN 175 includes sequences that playa role in the mortal-cell specific repression of hTRT gene expression isachieved. It will be recognized that less than the entire approximately120 basepair sequence may be required for this effect, and that othersequences not in the approximately 120 base pair region may also play arole (independently or in combination with the approximately 120 basepair region) in regulation of hTRT expression. Thus, the approximately120 base pair region includes all or part of one or more cis-actingelements.

[1065] Without intending to be bound by any particular mechanism, theapproximately 120 base pair sequence includes a binding site for arepressor (e.g., a trans acting repressor) which upon binding preventsinitiation of transcription of the hTRT gene. Such a repressor may bethe product of an anti-oncogene (e.g., a novel anti-oncogene), which canbe identified and cloned in accordance with the teachings herein and theuse of the novel reagents disclosed herein. In normal cells, repressorbinding or interaction with hTRT regulatory sequences (e.g., includingor within the approximately 120 base pair sequence) results in theabsence of hTRT protein and therefore of telomerase activity. Activationof telomerase in cancer cells can result from the loss of hTRT repressoractivity.

[1066] A number of applications of the “approximately 120 base pairregion” described above will be immediately apparent upon review of thisdisclosure, including for treatment or diagnosis of telomerase relateddiseases and identification of agents with telomerase modulatoryactivity. For example, using standard techniques, the sequence may beused to identify agents or proteins (e.g. naturally occurring repressorproteins) that specifically bind to the approximately 120 base pairsequence or a subsequence thereof. In addition, synthetic or naturallyoccurring agents that increase or stabilize repression (e.g., by bindingor otherwise interacting with the sequence, by stabilizing binding by anaturally occurring repressor, or by other means) will be useful forreducing telomerase activity in a cell (e.g., for treatment ofmalignancy). Similarly, agents that reduce repression (e.g., byinhibiting repressor binding, or by other means) will be useful forincreasing telomerase expression (e.g., by controlled activation), forexample to increase the proliferative capacity of normal cells).

Example 15 Subcellular Localization of hTRT

[1067] A fusion protein having hTRT and enhanced green fluorescentprotein (EGFP; Cormack et al., 1996, Gene 173:33) regions wasconstructed as described below. The EGFP moiety provides a detectabletag or signal so that the presence or location of the fusion protein canbe easily determined. Because EGFP-fusion proteins localize in thecorrect cellular compartments, this construct may be used to determinethe subcellular location of hTRT protein.

[1068] A. Construction of pGRN138

[1069] A vector for expression of an hTRT-EGFP fusion protein inmammalian cells was constructed by placing the EcoRI insert from pGRN124(see Example 6) into the EcoRI site of pEGFP-C2 (Clontech, San Diego,Calif.). The amino acid sequence of the fusion protein is providedbelow. EGFP residues are in bold, residues encoded by the 5′untranslated region of hTRT mRNA are underlined, and the hTRT proteinsequence is in normal font. MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEG (SEQ IDNO:628) EGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRH NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELY KSGRTQISSSSFEFAAAST QRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRL GPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAF GFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCA YQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLP KRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQH HAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPW MPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPE EEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQE LTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFF YRPSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGAR TFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKV DVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAH LQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYG DMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALG GTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASVTFNRGFKAGRNMRRKLFGVLRLKCH SLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKN AGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAA NPALPSDFKTILD

[1070] Other EGFP fusion constructs can be made using partial (e.g.,truncated) hTRT coding sequence and used, as described infra, toidentify activities of particular regions of the hTRT polypeptide.

[1071] B. Nuclear Localization and Uses of pGRN138

[1072] Transfection of NIH 293 and BJ cells with pGRN138 confirmed thenuclear localization of recombinantly expressed hTRT. Cells weretransfected with pGRN138 (EGFP-hTRT) and with a control construct(expressing EGFP only). Nuclear localization of the EGFP-hTRT isapparent in both cell types by fluorescence microscopy. As noted supra,the pGRN138 hTRT-GFP fusion protein supports reconstitution oftelomerase activity in both an in vitro transcription translation systemand in vivo when transfected into BJ cells.

[1073] The hTRT-EGFP fusion proteins (or similar detectable fusionproteins) can be used in a variety of applications. For example, thefusion construct described in this example, or a construct of EGFP and atruncated form of hTRT, can be used to assess the ability of hTRT andvariants to enter a cell nucleus and/or localize at the chromosome ends.In addition, cells stably or transiently transfected with pGRN138 areused for screening compounds to identify telomerase modulatory drugs orcompounds. Agents that interfere with nuclear localization or telomerelocalization can be identified as telomerase inhibitors. Tumor celllines stably expressing EGFP-hTRT can be useful for this purpose.Potential modulators of telomerase will be administered to thesetransfected cells and the localization of the EGFP-hTRT will beassessed. In addition, FACS or other fluorescence-based methods can beused to select cells expressing hTRT to provide homogeneous populationsfor drug screening, particularly when transient transfection of cells isemployed.

[1074] In other applications, regions of the hTRT can be mutagenized toidentify regions (e.g., residues 193-196 (PRRR; SEQ ID NO:541) and235-240 (PKRPRR; SEQ ID NO:542)) required for nuclear localization,which are targets for anti-telomerase drugs (telomerase activitymodulators). Other applications include:

[1075] use of the fusion protein as a fluorescent marker of efficientcell transfection for both transient transfection experiments and whenestablishing stable cell lines expressing EGFP-hTRT;

[1076] expression of an hTRT-EGFP fusion with mutated nuclearlocalization signals (deficient for nuclear localization) in immortalcells so that the hTRT mutant-EGFP scavenges all the hTR of the immortalcells, retaining it in the cytoplasm and preventing telomeremaintenance; and

[1077] use as a tagged protein for immunoprecipitation.

Example 16 Effect of Mutation on Telomerase Catalytic Activity

[1078] This example describes hTRT variant proteins having altered aminoacids and altered telomerase catalytic activity. Amino acidsubstitutions followed by functional analysis is a standard means ofassessing the importance and function of a polypeptide sequence. Thisexample demonstrates that changes in the reverse transcriptase (RT) andtelomerase (T) motifs affect telomerase catalytic activity.

[1079] Conventional nomenclature is used to describe mutants: the targetresidue in the native molecule (hTRT) is identified by one-letter codeand position, and the corresponding residue in the mutant protein isindicated by one-letter code. Thus, for example, “K626A” specifies amutant in which the lysine at position 626 (i.e., in motif 1) of hTRT ischanged to an alanine.

[1080] A. Mutation of hTRT FFYxTE (SEQ ID NO:360) Motif

[1081] In initial experiments, a vector encoding an hTRT mutant protein,“F560A,” was produced in which amino acid 560 of hTRT was changed fromphenylalanine (F) to alanine (A) by site directed mutagenesis of pGRN121using standard techniques. This mutation disrupts the TRT FFYxTE (SEQ IDNO:360) motif. The resulting F560A mutant polynucleotide was shown todirect synthesis of a full length hTRT protein as assessed using acell-free reticulocyte lysate transcription/translation system in thepresence of ³⁵S-methionine.

[1082] When the mutant polypeptide was co-translated with hTR, asdescribed in Example 7, no telomerase activity was detected as observedby TRAP using 20 cycles of PCR, while a control hTRT/hTR cotranslationdid reconstitute activity. With 30 cycles of PCR in the TRAP assay,telomerase activity was observable with the mutant hTRT, but wasconsiderably lower than the control (wild-type) hTRT.

[1083] B. Additional Site-Directed Mutagenesis of hTRT Amino AcidResidues

[1084] Conserved amino acids in six RT motifs were changed to alanineusing standard site directed mutagenesis techniques (see, e.g., Ausubel,supra) to assess their contribution to catalytic activity. The mutantswere assayed using IVR telomerase using the two step conventional/TRAPassay detailed in example 7.

[1085] The K626A (motif 1), R631A (motif 2), D712A (motif A), Y717A(motif A), D868A (motif C) mutants had greatly reduced or undetectabletelomerase activity (<1% of wild-type), while the Q833A (motif B) andG932A (motif E) mutants exhibited low/intermediate levels of activity(<10% of wild-type). Two mutations outside the RT motifs, R688A andD897A, had activity equivalent to wild type hTRT. These results wereconsistent with analogous mutations in reverse transcriptases (Joyce etal., 1994, Ann. Rev. Biochem. 63:777) and are similar to resultsobtained with Est2p (see Lingner, 1997, Science 276:561). Theexperiments identify residues in the RT motifs critical and not criticalfor enzymatic activity and demonstrate that hTRT is the catalyticprotein of human telomerase. The mutations provide variant hTRTpolypeptides that have utility, e.g., as dominant/negative regulators oftelomerase activity.

[1086] Amino acid alignment of the known TRTs identified atelomerase-specific motif, motif T (see supra). To determine thecatalytic role of this motif in hTRT, a six amino acid deletion in thismotif (Δ560-565; FFYxTE; SEQ ID NO:360), was constructed using standardsite directed mutagenesis techniques (Ausubel, supra). The deletion wasassayed using IVR telomerase using the two step conventional/TRAP assaydetailed in Example 7. The Δ560-565 mutant had no observable telomeraseactivity after 25 cycles of PCR whereas wild type hTRT IVR telomeraseproduced a strong signal. Each amino acid in each residue in motif T wasexamined independently in a similar manner; mutants F560A, Y562A, T564A,and E565A retained intermediate levels of telomerase activity, while acontrol mutant, F487A, had minimal affect on activity. Notably, mutantF561A had greatly reduced or undetectable telomerase activity, whileactivity was fully restored in its “revertant”, F561A561F. F561A561Fchanges the mutated position back to its original phenylalanine. This isa control that demonstrates that no other amino acid changes occurred tothe plasmid that could account for the decreased activity observed.Thus, the T motif is the first non-RT motif shown to be absolutelyrequired for telomerase activity.

[1087] Motif T can be used for identification of TRTs from otherorganisms and hTRT proteins comprising variants of this motif can beused as a dominant/negative regulator of telomerase activity. Unlikemost other RTs, telomerase stably associates with and processivelycopies a small portion of a single RNA (ie. hTR), thus motif T can beinvolved in mediating hTR binding, the processivity of the reaction, orother functions unique to the telomerase RT.

[1088] In other experiments, it was observed that the deletion variantencoded by pro90hTRT described herein, did not reconstitute telomeraseactivity when co-synthesized with hTR, as measured using a modified TRAPassay (Autexier et al., 1996, EMBO Journal 15:5928, which isincorporated herein by reference).

Example 17 Screening for Telomerase Activity Modulators UsingRecombinantly Expressed Telomerase Components

[1089] This example describes the use of in vitro reconstitutedtelomerase for screening and identifying telomerase activity modulators.The assay described is easily adapted to high-through-put methods (e.g.,using multiple well plates and/or robotic systems). Numerous variationson the steps of the assay will be apparent to one of skill in the artafter review of this disclosure.

[1090] Recombinant clones for telomerase components (e.g., hTRT and hTR)are transcribed and translated (hTRT only) in an in vitro reaction asfollows and as described in Example 7 supra, using the TNT7 T7 CoupledReticulocyte lysate system (Promega), which is described in U.S. Pat.No. 5,324,637, following the manufacturer's instructions: Reagent Amountper reaction (μL) TNT Rabbit Reticulocyte lysate 25 TNT reaction buffer2 TNT T7 RNA Pol. 1 AA mixture (complete) 1 Prime RNase inhibitor 1Nuclease-free water 16 Xba1 cut pGRN121 [hTRT] (0.5 μg) 2 Fsp1 cutpGRN164 [hTR] (0.5 μg) 2

[1091] The reaction is incubated at 30° C. for 2 hours. The product isthen purified on an ultrafree-MC DEAE filter (Millipore).

[1092] The recombinant telomerase product (IVRP) is assayed in thepresence and absence of multiple concentrations of test compounds whichare solubilized in DMSO (e.g. 10 μM-100 μM). Test compounds arepreincubated in a total volume of 25 μL for 30 minutes at roomtemperature in the presence of 2.5 μL IVRP, 2.5% DMSO, and 1×TRAP Buffer(20 mM Tris-HCl, pH 8.3, 1.5 mM MgCl₂, 63 mM KCl, 0.05% Tween20, 1.0 mMEGTA, 0.1 mg/ml Bovine serum albumin). Following the preincubation, 25μL of the TRAP assay reaction mixture is added to each sample. The TRAPassay reaction mixture is composed of 1×TRAP buffer, 50 μL dNTP, 2.0μg/ml primer ACX, 41 g/ml primer U2, 0.8 attomol/ml TSU2, 2 units/50 μlTaq polymerase (Perkin Elmer), and 2 μg/ml [³²P]5′end-labeled primer TS(3000 Ci/mmol). The reaction tubes are then placed in the PCRthermocycler (MJ Research) and PCR is performed as follows: 60 min at30° C., 20 cycles of {30 sec at 94° C., 30 sec. at 60° C., 30 sec. at72° C.}, 1 min at 72° C., cool down to 10° C. The TRAP assay isdescribed, as noted supra, in U.S. Pat. No. 5,629,154. The primers andsubstrate used have the sequences: TS Primer (5′-AATCCGTCGAGCAGAGTT-3′;SEQ ID NO:629); ACX Primer (5′-GCGCGG[CTTACC]₃CTAACC-3′; SEQ ID NO:630);U2 primer (5′-ATCGCTTCTCGGCCTTTT-3′; SEQ ID NO:631); TSU2(5′-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3′; SEQ ID NO:632)

[1093] After completion of the PCR step, 4 μl of 10× loading buffercontaining bromophenol blue is added to each reaction tube and products(20 μl) are run on a 12.5% non-denaturing PAGE in 0.5×TBE at 400 V. Thecompleted gel is subsequently dried and the TRAP products are visualizedby Phosphorimager or by autoradiography. The telomerase activity in thepresence of the test compound is measured by comparing the incorporationof label in reaction product to a parallel reaction lacking the agent.

[1094] The following clones described in the Examples have beendeposited with the American Type Culture Collection, Rockville, Md.20852, USA: Lambda phage λ 25-1.1 ATCC accession number 209024 pGRN121ATCC accession number 209016 Lambda phage λGΦ5 ATCC accession number98505

[1095] The present invention provides novel methods and materialsrelating to hTRT and diagnosis and treatment of telomerase-relateddiseases. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Many variations of theinvention will become apparent to those of skill in the art upon reviewof this specification. The scope of the invention should, therefore, bedetermined not with reference to the above description, but insteadshould be determined with reference to the appended claims along withtheir full scope of equivalents.

[1096] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040247613). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed is:
 1. A method for treating cancer in a human subject,comprising administering to the subject a vaccine comprising animmunogenic polypeptide having an amino acid sequence of 5 or moreconsecutive amino acids of SEQ. ID NO:2, or a polynucleotide encodingsaid amino acid sequence, whereby administration of the vaccine to ahuman subject elicits a specific immune response against humantelomerase reverse transcriptase (hTRT).
 2. A method for treating cancerin a human subject, comprising administering to the subject a vaccinecomprising an immunogenic polypeptide having an amino acid sequence of10 or more consecutive amino acids of SEQ. ID NO:2, with or without aconservative amino acid substitution, or a polynucleotide encoding saidamino acid sequence, whereby administration of the vaccine to a humansubject elicits a specific immune response against human telomerasereverse transcriptase (hTRT).
 3. A method for treating cancer in a humansubject, comprising administering to the subject a vaccine comprising animmunogenic polypeptide having an amino acid sequence of 5 or moreconsecutive amino acids of SEQ. ID NO:2 fused to a sequence of anotherprotein, or a polynucleotide encoding said amino acid sequence, wherebyadministration of the vaccine to a human subject elicits a specificimmune response against human telomerase reverse transcriptase (hTRT).4. The method of claim 1, wherein the amino acid sequence contains 15 ormore consecutive amino acids of SEQ. ID NO:2.
 5. The method of claim 1,wherein the amino acid sequence contains 100 or more consecutive aminoacids of SEQ. ID NO:2.
 6. The method of claim 1, wherein the amino acidsequence contains Motif T (SEQ. ID NO:350 or 351).
 7. The method ofclaim 1, wherein the amino acid sequence contains Motif 1 (SEQ. IDNO:355 or 633); Motif 2 (SEQ. ID NO:356 OR 634); Motif A (SEQ. ID NO:357or 635); Motif B′ (SEQ. ID NO:358 OR 636); and Motif C (SEQ. ID NO:359or 637).
 8. The method of claim 1, wherein said amino acid sequence isthe sequence of full length hTRT (SEQ. ID NO:2).
 9. The method of claim1, wherein said polypeptide has telomerase catalytic activity whencomplexed with telomerase RNA component.
 10. The method of claim 1,wherein said polypeptide contains at least one mutation or deletion thatreduces or eliminates telomerase catalytic activity.
 11. The method ofclaim 10, wherein said mutation or deletion is in Motif 1, Motif 2,Motif A, or Motif C.
 12. The method of claim 10, wherein said mutationor deletion is in Motif T.
 13. The method of claim 10, wherein saidpolypeptide contains a deletion of the motif FFYxTE (SEQ. ID NO:360).14. The method of claim 1, wherein a chemically synthesized polypeptidecontaining said amino acid sequence is formulated as the vaccine. 15.The method of claim 1, wherein a viral vector encoding said amino acidsequence is formulated as the vaccine.
 16. The method of claim 15,wherein an expression vector is formulated as the vaccine, wherein theregion encoding said polypeptide is under control of a promoter for aviral or mammalian gene.
 17. The method of claim 15, wherein the vectoris an adenovirus vector.
 18. The method of claim 1, wherein the vaccineis formulated to contain an immunological adjuvant.
 19. A method fortreating cancer in a human subject, comprising administering to thesubject a vaccine comprising an immunogenic polypeptide having an aminoacid sequence of 5 or more consecutive amino acids of SEQ. ID NO:2, or apolynucleotide encoding said amino acid sequence, whereby administrationof the vaccine to a human subject having cancer is effective in treatingthe cancer.
 20. The method of claim 19, wherein the cancer is prostatecancer.